Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes.

BACKGROUND: Securing a diagnosis of ovarian cancer and establishing means to predict outcomes to therapeutics remain formidable clinical challenges. Early diagnosis is particularly important since survival rates are markedly improved if tumour is detected early. METHODS: Comprehensive miRNA profiles were generated on presurgical plasma samples from 42 women with confirmed serous epithelial ovarian cancer, 36 women diagnosed with a benign neoplasm, and 23 comparably age-matched women with no known pelvic mass. RESULTS: Twenty-two miRNAs were differentially expressed between healthy controls and the ovarian cancer group (P<0.05), while a six miRNA profile subset distinguished presurgical plasma from benign and ovarian cancer patients. There were also significant differences in miRNA profiles in presurgical plasma from women diagnosed with ovarian cancer who had short overall survival when compared to women with long overall survival (P<0.05). CONCLUSION: Our preliminary data support the utility of circulating plasma miRNAs to distinguish women with ovarian cancer from those with a benign mass and identify women likely to benefit from currently available treatment for serous epithelial ovarian cancer from those who may not.

The hedgehog pathway as a therapeutic target for treatment of breast cancer.

The Hedgehog (Hh) signaling pathway plays a key role in a variety of processes, such as embryogenesis and maintenance of adult tissue homeostasis. It is also becoming increasingly clear that this pathway can have a crucial role in tumorigenesis. Most recently, the Hh signaling pathway has been implicated in the development and maintenance of breast cancer. Here we review Hh signaling, advances in small molecule and antibody-based inhibitors targeting the Hh pathway, and dysregulation of the Hh signaling pathway in breast cancer.

The heat shock protein 90 chaperone complex: an evolving therapeutic target.

Hsp90 (heat shock protein 90) is a molecular chaperone that modulates the stability and/or transport of a diverse set of critical cellular regulatory, metabolism, organization, and signaling proteins. Binding to Hsp90 is required for normal function of many proteins. In addition, Hsp90 has an extra-cellular function. It is found in two isotypes: alpha which is inducible and beta which is constitutive. Tumor cells frequently over express Hsp90alpha, and Hsp90 is implicated in cancer progression. Hence Hsp90 has emerged as a potential target for cancer treatment. A variety of agents have been found to interfere with Hsp function, mainly by binding to an ATP binding site on the molecule. More recent agents interfere with protein binding or the dimerization of Hsp90 needed for function. Preclinical studies have demonstrated that disruption of the many client proteins chaperoned by Hsp90 is achievable and associated with significant growth inhibition, both in vitro and in tumor xenografts. As a result, agents which interfere with this protein's function are being tested in the clinic as a targeted method of interfering with malignant growth. We review the current clinical status of therapeutic efforts to perturb this pathway and discuss future directions.

Implications of applied research for prognosis and therapy of breast cancer.

Breast cancer is the one of leading causes of cancer-related deaths in women within economically developed regions of the world. The heterogeneity of the natural history of breast cancer complicates patient management in that there is tremendous variability in response to treatment and for survival. More recently, several biomarkers (hormone receptor status and HER2 expression) have been added to the risk evaluation and therapeutic assessments. Evolving knowledge of molecular biology and newer techniques, such as genomics and proteomics, offer the potential to better define the biologic nature of the disease process, both for risk and therapy. This review discusses classical as well as new prognostic and predictive techniques. These are leading to a paradigm shift from empirical treatment to an individually tailored approach, which may soon become a realistic option for patients, based on specific molecular profiles.

Heparin-induced thrombocytopenia complicating hemodialysis.

Hemodialysis complicated by heparin-induced thrombocytopenia (HIT) is a rare event requiring anticoagulation with direct-thrombin inhibitors. Contaminant calcific uremic arteriolopathy (calciphylaxis) further complicates this situation due to the possibility that warfarin anticoagulation may exacerbate skin necrosis. The authors report a patient with renal failure and calciphylaxis who developed HIT after starting hemodialysis. She was successfully treated with Argatroban.

A phase I and pharmacokinetic study of irinotecan in patients with hepatic or renal dysfunction or with prior pelvic radiation: CALGB 9863.

BACKGROUND: To ascertain if hepatic or renal dysfunction or prior pelvic radiation (XRT) leads to increased toxicity at a given dose of irinotecan and to characterize the pharmacokinetics of irinotecan and its major metabolites in patients with hepatic or renal dysfunction. PATIENTS AND METHODS: Adults with tumors appropriate for irinotecan therapy and who had abnormal liver or renal function tests or had prior radiation to the pelvis were eligible. Patients were assigned to one of four treatment cohorts: I, aspartate aminotransferase (AST) > or = 3x upper limit of normal and direct bilirubin <1.0 mg/dl; II, direct bilirubin 1.0-7.0 mg/dl; III, creatinine 1.6-5.0 mg/dl with normal liver function; IV, prior pelvic XRT with normal liver and renal function. Starting with reduced doses of either 145 or 225 mg/m(2), irinotecan was administered every 3 weeks to at least three patients within each cohort. Irinotecan and its metabolites in the blood were measured in all patients. RESULTS: Thirty-five patients were evaluable for toxicity. No dose-limiting toxicity was seen in cohort I, although only three patients were treated and at a dose of 225 mg/m(2). Patients with elevations of direct bilirubin had dose-limiting toxicities, even though the starting dose was 145 mg/m(2). These same patients appeared to have comparable exposure to the active metabolite SN-38 as normal patients treated with full-dose irinotecan. Patients with elevations of creatinine or with prior pelvic radiotherapy did not appear to have increased risk of toxicity at the doses explored in this study. CONCLUSIONS: Patients with elevated bilirubin treated with irinotecan have an increased risk of toxicity and a dose reduction is recommended. Patients with elevated AST, creatinine or prior pelvic radiation do not appear to have increased sensitivity to irinotecan, but the data are not adequate to support a specific dosing recommendation.

Unique induction of p21(WAF1/CIP1)expression by vinorelbine in androgen-independent prostate cancer cells.

To study the mechanisms of the development of hormone refractory prostate cancer, we established an androgen-independent (AI) prostate cancer cell line derived from hormone-dependent (AD) LNCaP cells. Our previous studies have demonstrated that AI cells are deficient in expression of p21(WAFl/CIP1) (p21) due to overexpressed AR and are resistant to apoptosis. In this study, the induction of p53 and p21 expression by vinorelbine (Navelbine) was compared between AD and AI cells in an attempt to understand the difference(s) in apoptotic signalling pathways in these cells. Using a series of deletion of p21 reporter constructs, we found that vinorelbine mediated p21 induction in a p53-dependent manner in AD cells. In contrast, p21 expression restored by vinorelbine in AI cells was found to be through both p53-dependent and-independent pathways. In the absence of two p53 binding sites, Spl-3 and Spl-4 sites, in the promoter of human p21 gene, were found to be required for vinorelbine-mediated p21 activation. No p21 induction was observed by paclitaxel in AI cells. Exposure of AI cells to paciltaxel followed by vinorelbine produced synergism. Our data, thus, provide a basis for the synergistic combination of vinorelbine and paclitaxel for the treatment of advanced prostate cancer.

Comparable efficacy and safety profiles of once-per-cycle pegfilgrastim and daily injection filgrastim in chemotherapy-induced neutropenia: a multicenter dose-finding study in women with breast cancer.

BACKGROUND: Neutropenia is common in patients receiving myelotoxic chemotherapy. Pegfilgrastim, a sustained-duration filgrastim is a once-per-cycle therapy for prophylactic neutrophil support. PATIENTS AND METHODS: Women, treated with four cycles of doxorubicin/docetaxel chemotherapy every 21 days, received pegfilgrastim or filgrastim 24 h after chemotherapy as a single subcutaneous injection per chemotherapy cycle (pegfilgrastim 30, 60 or 100 microg/kg) or daily subcutaneous injections (filgrastim 5 microg/kg/day). Safety, efficacy and pharmacokinetics were analyzed. RESULTS: The incidence of grade 4 neutropenia in cycle 1 was 95, 90 and 74%, in patients who received pegfilgrastim 30, 60 and 100 microg/kg, respectively, and 76% in patients who received filgrastim. Mean duration of grade 4 neutropenia in cycle 1 was 2.7,2 and 1.3 days for doses of pegfilgrastim, and 1.6 days for filgrastim. The pharmacokinetics of pegfilgrastim were non-linear and dependent on both dose and neutrophil count. Pegfilgrastim serum concentration was sustained until the neutrophil nadir occurred then declined rapidly as neutrophils started to recover, consistent with a self-regulating neutrophil-mediated clearance mechanism. The safety profiles of pegfilgrastim and filgrastim were similar. CONCLUSIONS: A single subcutaneous injection of pegfilgrastim 100 microg/kg provided neutrophil support and a safety profile comparable to daily subcutaneous injections of filgrastim during multiple chemotherapy cycles.

Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells.

The effects of vinorelbine and paclitaxel on the activity of extracellular signal-regulated protein kinase2 (ERK2), a member of MAP kinase, and its role in the induction of bcl-2 phosphorylation and apoptosis were evaluated in MCF-7 cells. We demonstrated that ERK2 was activated rapidly by vinorelbine, and was inhibited by either paclitaxel or estramustine. A 3-fold increase of ERK2 kinase activity was observed within 30 min when MCF-7 cells were treated with 0.1 microM vinorelbine. In contrast, the same treatment with paclitaxel resulted in a significant decrease of ERK2 kinase activity. We also demonstrated that elevated bcl-2 phosphorylation induced by vinorelbine is paralleled by decrease of a complex formation between bcl-2 and bax, cleavage of poly (ADP) ribose polymerase (PARP) protein, activation of caspase-7, and apoptosis. The levels of bcl-2 phosphorylation, bax, and PARP were not significantly affected by 2'-amino-3'-methoxyflavone (PD 98059), an ERK kinase specific inhibitor. Thus, our data suggest that the apoptosis induced by vinorelbine in MCF-7 cells is mediated through the bcl-2 phosphorylation/bax/caspases pathways, and that activation of ERK2 by vinorelbine does not directly lead to the drug-mediated apoptosis. Since decrease of PARP occurred quickly following the treatment of MCF-7 cells with either 0.1 microM of vinorelbine or paclitaxel, this protein may serve as an early indicator of apoptosis induced not only by DNA damaging agents, but also by antimicrotubule drugs.

In vitro effects of dexrazoxane (Zinecard) and classical acute leukemia therapy: time to consider expanded clinical trials?

Anthracyclines have been the backbone of acute leukemia therapy in the adult for many years, but little attention has been paid to the long-term toxicity of these agents in this disease because of the poor survival of this population of patients. Recent studies have examined dose-intensified daunorubicin with dosages as high as 95 mg/m2 daily x 3 in this population with the attendant concerns of both acute and chronic toxicity. We have examined three human leukemia cell lines in vitro, treated with either daunorubicin, mitoxantrone, with or without cytosine arabinoside in the presence of dexrazoxane to determine whether such treatment would be synergistic or antagonistic. AML-193, CRF-SB, and Molt-4 cell lines were grown to confluence, plated into microtiter dishes and incubated for 72 h with varying concentrations of the above drugs. Cytotoxicity was determined by the MTT assay, and synergy or antagonism by median effect analysis. Dexrazoxane demonstrated additive or synergistic cytotoxic effects (CI <1) under most conditions. The triplet of daunorubicin, cytosine arabinoside, and dexrazoxane showed profound synergy in all three cell lines. These effects occurred at clinically achievable levels. If high dosages of anthracyclines are contemplated in this population, these preclinical data suggest that the addition of dexrazoxane to classical therapy is not antagonistic and thus may allow an investigation of the role of dexrazoxane as a cardiac protectant.

Treatment of aggressive non-Hodgkin's lymphoma in elderly patients with thiotepa, Novantrone (mitoxantrone), vincristine, prednisone (TNOP).

The aging of the population and the increased incidence of non-Hodgkin's lymphoma will result in a large number of elderly patients with this disorder. Newer therapies will be required for this group of patients. This article reports a new therapy for elderly patients with diffuse aggressive non-Hodgkin's lymphoma. Patients were treated with TNOP (thiotepa 20 mg/m(2), mitoxantrone (Novantrone) 10 mg/m(2), vincristine (Oncovin) 1 mg/m(2) all on day 1 and prednisone 60 mg/m(2) on days 1 through 5 of a 21-day course. Twenty-six patients were enrolled on study. The patients' ages ranged from 66 years to 87 years, with a mean age of 75.5 years. Eleven patients had a partial response (42%) and 4 patients had a complete response (15%) for a total response of 57%. Eighty-one percent of patients survived 1 year and 54% survived for 2 years. The median survival was 26 months. Hematologic and nonhematologic toxicity was tolerable. We believe that TNOP is an excellent therapeutic option in this group of elderly patients, particularly in the palliative setting.

Phase I and pharmacokinetic study of LY309887: a specific inhibitor of purine biosynthesis.

PURPOSE: In this phase I trial in humans the safety and pharmacology of LY309887 on a weekly schedule combined with daily oral 5-mg doses of folic acid were evaluated. BACKGROUND: LY309887 is an inhibitor of folate-dependent enzymes involved in de novo purine biosynthesis and has a broad preclinical antitumor activity. In murine systems, combining this agent with exogenous folic acid results in an enhanced therapeutic index. METHODS: This study was a single-institution, open-label, clinical trial of dose escalation with toxicity and pharmacokinetic parameters determined. The dose range studied was 0.5-4 mg/m2 per week x6 and then a modified schedule weekly x3 every 6 weeks. RESULTS: Dose-limiting toxicities were of delayed onset and associated with hematologic, neurologic, and mucosal effects. Pharmacokinetic parameters revealed dose linearity for AUC and Cmax. Low circulating levels of drug persisted for over 200 h. Urinary excretion accounted for approximately 50% of the parent drug but was highly variable. The urinary excretion was near maximal within 24 h of dosing. CONCLUSIONS: The modified dosing schedule allowed repetitive dosing in patients. Further evaluation of the 2 mg/m2 per week x3 every 6 weeks with daily oral folate supplement as a potential phase II dose may be warranted.

Social support as a buffer to the psychological impact of stressful life events in women with breast cancer.

BACKGROUND: Three theoretical models by which social support may influence the impact of stressful life events on cancer patients' psychological state were tested: 1) the additive model, in which social support and stressful life events each directly influence cancer patients' adjustment, irrespective of the magnitude of the other; 2) the buffering hypothesis, in which stressful events occurring in the presence of social support should produce less distress than if they occur in its absence; and 3) both additive and buffering models. METHODS: One hundred seventy-nine patients who had Stage II breast cancer (median age, 56 yrs; 68% disease free), treated a mean of 6.8 years since entry to Cancer and Leukemia Group B (CALGB) 8541, were interviewed by telephone concerning their psychosocial adjustment. The following measures were used: Medical Outcome Study Social Support Survey (MOS-SSS), Life Experience Survey (LES) a measure of stressful life events within the past 12 months, European Organization for Research on the Treatment of Cancer (EORTC QLQ-C30) a measure of quality of life, Mental Health Inventory (MHI), and the Systems of Belief Inventory (SBI) a measure of spiritual and religious involvement. RESULTS: Hierarchical regression analyses revealed that less than excellent levels of social support (P < 0.01), greater negative impact of LES fateful life events (e.g., death of family member) (P < 0.05), personal illness or injury (P < 0.05), and all other negative life events in the past year (< 4; P < 0.01) were significant predictors of greater MHI psychological distress, in addition to being divorced or separated (P < 0.001), and more recently treated for cancer on CALGB 8541 (P < 0.05). The interaction of LES scores with MOS-SSS or SBI social support, used to test the buffering hypothesis, did not significantly improve the prediction of MHI psychological distress. CONCLUSIONS: The results supported the additive model, with both stressful life events and social support independently and significantly affecting patients' emotional state. However, the level of social support needed to be very high to reduce the likelihood of severe psychological distress.

A reexamination of PSC 833 (Valspodar) as a cytotoxic agent and in combination with anticancer agents.

BACKGROUND: The cyclosporins have been thought as being mainly immunosuppressive agents which interfere with the function of the MDR pump and thus play a role in resistance to drug anticancer effects. We reexamined their cytotoxicity in defined cell lines both as single agents and in combination with agents which may be of value in human malignant disease. METHODS: Cells were grown to confluence following inoculation at 5,000-8,000 cells/well in 96-well dishes, and growth patterns and death were determined by an MTT assay. Median effect analysis was used to look for synergy, additive effects, or antagonism between the cyclosporins and drugs with antitumor effects in humans. RESULTS: Cyclosporin A and PSC 833 were found to have cytotoxic activity at clinically achievable concentrations in breast, leukemia, and prostate cell lines. Synergistic or additive effects were demonstrated in all three prostate cell lines when PSC 833 was combined with estramustine, etoposide, ketoconazole, suramin, or vinorelbine in the prostate cancer cell lines. Cell line-selective additive effects or synergism were also identified with bicalutamide, carboplatin, cisplatinum, cis-retinoic acid, dexamethasone, 5-fluorouracil, liarozole, and trans-retinoic acid. CONLCLUSIONS: PSC 833 or cyclosporin alone or in combination with other agents may have an anticancer effect independently of their modulatory action on MDR. Several of the synergistic combinations which are not mediated by the MDR pump need to be tested in vivo for efficacy.

Synergism of cytotoxic effects of vinorelbine and paclitaxel in vitro.

Empiric combinations of vinca alkaloids with taxanes have been recently used in clinical oncology. To enhance the activity of these two classes of agents, we evaluated the sequence and duration of exposure, looking for synergistic effects. Cell lines DU 145, PC 3, LnCaP, LL 86, MCF7wt, and MCF7/ADR (NCI/ADR-RES) were incubated with varying concentrations of paclitaxel or vinorelbine. Cytotoxicity was evaluated by a semiautomated MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) method. Synergism or antagonism of these two agents either sequentially or in combination was determined by median effect analysis. Prolonged exposure of cells to either drug enhanced cytotoxic effect. Synergism or antagonism with vinorelbine and paclitaxel were both sequence dependent and cell line specific. In the case of MCF7wt, synergism was seen when a 48-hr exposure to vinorelbine preceded paclitaxel, whereas antagonism was noted when both agents were applied simultaneously or when the sequence was reversed. Concurrent vinorelbine and paclitaxel were synergistic in four of six cell lines when the exposure was extended to 96 hr but not for shorter durations of exposure. Sequential exposure of vinorelbine preceding paclitaxel or prolonged exposure to both agents concurrently needs to be tested clinically to determine whether the antitumor activity of this combination can be enhanced. In addition, these studies suggest concurrent administration of these two agents may lead to a less than optimal cytotoxic result.

Capecitabine.

Drug development has undergone rapid shifts in methodology and the use of rationally derived agents which either target specific tissues or molecules such as receptors or enzymatic sites. Capecitabine is a rationally derived prodrug of 5-fluorouracil which is based upon the known high concentration of the enzyme thymidine phosphorylase in many human tumors. The first prodrug designed to exploit this biochemical finding was 5-DFUR which allowed cytotoxic 5-fluorouracil to be preferentially concentrated in tumors. Unfortunately, in man this agent was associated with significant gastrointestinal toxicity. Further manipulation of this molecular resulted in capecitabine which is a relatively inert prodrug, undergoes three enzymatic steps, and offers the potential of less gastrointestinal toxicity. Phase I trials have examined several schedules with the divided oral daily x 14 schedule every 3 weeks as the preferred phase II and phase III dosing method. This agent demonstrates significant antitumor effect in diseases known to be responsive to fluoropyrimidines. Further study is needed to determine whether capecitabine has a broader spectrum of action thus affecting other tumor types than 5-fluorouracil. Major dose limiting toxicities have been hand foot syndrome, nausea/vomiting, and diarrhea.

HER-2/neu and p53 expression versus tamoxifen resistance in estrogen receptor-positive, node-positive breast cancer.

PURPOSE: An association between the overexpression of proto-oncogene HER-2/neu and resistance to tamoxifen in estrogen receptor (ER)-positive primary and metastatic breast cancer has been suggested. We examine a possible interaction between HER-2/neu or p53 expression and tamoxifen effectiveness in patients with ER-positive, node-positive disease treated with cyclophosphamide, doxorubicin, and fluorouracil in a large adjuvant chemotherapy trial (Cancer and Leukemia Group B [CALGB] 8541). Tamoxifen assignment was not randomized-physician discretion was used for premenopausal and postmenopausal women. Trial protocol then specified assignment to postmenopausal women with ER-positive tumors, although not all took tamoxifen. PATIENTS AND METHODS: CALGB 8541 assessed HER-2/neu expression in patients with ER-positive disease by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) and amplification by differential polymerase chain reaction (PCR). IHC assessed expression of p53. Univariate and multivariate proportional hazards models assessed tamoxifen-HER-2/neu status interactions and tamoxifen-p53 status interactions. RESULTS: HER-2/neu status was available for 651 patients with ER-positive disease; 650, 608, and 353 patients were assessed by IHC, PCR, and FISH, respectively. Approximately one half received tamoxifen. Reduction in risk of disease recurrence or death resulting from tamoxifen was approximately 37% (32% with overexpression and 39% with normal expression of HER-2/neu; n = 155 by IHC). The tamoxifen-HER-2/neu status interaction was not significant in multivariate analysis of all three HER-2/neu assessment methods. Tamoxifen-p53 interaction did not significantly predict outcome. CONCLUSION: Disease-free and overall survival benefit of tamoxifen in patients with ER-positive, node-positive breast cancer does not depend on HER-2/neu or p53 status. Our data suggest that neither HER-2/neu nor p53 expression should be used to determine assignment of tamoxifen.

Rab6c, a new member of the rab gene family, is involved in drug resistance in MCF7/AdrR cells.

A new Rab6 homolog cDNA, Rab6c, was discovered by a hypermethylated DNA fragment probe that was isolated from a human multidrug resistant (MDR) breast cancer cell line, MCF7/AdrR, by the methylation sensitive-representational difference analysis (MS-RDA) technique. Rab6c was found to be under-expressed in MCF7/AdrR and MES-SA/Dx5 (a human MDR uterine sarcoma cell line) compared with their non-MDR parental cell lines. MCF7/AdrR cells expressing the exogenous Rab6c exhibited less resistance to several anti-cancer drugs, such as doxorubicin (DOX), taxol, vinblastine, and vincristine, than the control cells containing the empty vector. Flow cytometry experiments confirmed that the transfectants' diminished resistance to DOX was caused by increased drug accumulation induced by the exogenous Rab6c. These results indicate that Rab6c is involved in drug resistance in MCF7/AdrR cells.

Phase I and pharmacokinetic trial of gemcitabine in patients with hepatic or renal dysfunction: Cancer and Leukemia Group B 9565.

PURPOSE: To ascertain if hepatic or renal dysfunction leads to increased toxicity at a given dose of gemcitabine and to characterize the pharmacokinetics of gemcitabine and its major metabolite in patients with such dysfunction. PATIENTS AND METHODS: Adults with tumors appropriate for gemcitabine therapy and who had abnormal liver or renal function tests were eligible. Patients were assigned to one of three treatment cohorts: I-AST level less than or equal to two times normal and bilirubin level less than 1.6 mg/dL; II-bilirubin level 1.6 to 7.0 mg/dL; and III-creatinine level 1.6 to 5.0 mg/dL with normal liver function. Doses were explored in at least three patients within each cohort. Gemcitabine and its metabolite were to be measured in the blood in all patients. RESULTS: Forty patients were assessable for toxicity. Transient transaminase elevations were observed in many patients but were not dose limiting. Patients with AST elevations tolerated gemcitabine without increased toxicity, but patients with elevated bilirubin levels had significant deterioration in liver function after gemcitabine therapy. Patients with elevated creatinine levels had significant toxicity even at reduced doses of gemcitabine, including two instances of severe skin toxicity. There were no apparent pharmacokinetic differences among the three groups or compared with historical controls. CONCLUSION: If gemcitabine is used for patients with elevations in AST level, no dose reduction is necessary. Patients with elevated bilirubin levels have an increased risk of hepatic toxicity, and a dose reduction is recommended. Patients with elevated creatinine levels seem to have increased sensitivity to gemcitabine, but the data are not adequate to support a specific dosing recommendation.