Phase II study of MLN8237 (Alisertib) in advanced/metastatic sarcoma.

BACKGROUND: Aurora kinase A (AURKA) is commonly overexpressed in sarcoma. The inhibition of AURKA by shRNA or by a specific AURKA inhibitor blocks in vitro proliferation of multiple sarcoma subtypes. MLN8237 (alisertib) is a novel oral adenosine triphosphate-competitive AURKA inhibitor. PATIENTS AND METHODS: This Cancer Therapy Evaluation Program-sponsored phase II study of alisertib was conducted through the Alliance for Clinical Trials in Oncology (A091102). Patients were enrolled into histology-defined cohorts: (i) liposarcoma, (ii) leiomyosarcoma, (iii) undifferentiated sarcoma, (iv) malignant peripheral nerve sheath tumor, or (v) other. Treatment was alisertib 50 mg PO b.i.d. d1-d7 every 21 days. The primary end point was response rate; progression-free survival (PFS) was secondary. One response in the first 9 patients expanded enrollment in a cohort to 24 using a Simon two-stage design. RESULTS: Seventy-two patients were enrolled at 24 sites [12 LPS, 10 LMS, 11 US, 10 malignant peripheral nerve sheath tumor (MPNST), 29 Other]. The median age was 55 years; 54% were male; 58%/38%/4% were ECOG PS 0/1/2. One PR expanded enrollment to the second stage in the other sarcoma cohort. The histology-specific cohorts ceased at the first stage. There were two confirmed PRs in the other cohort (both angiosarcoma) and one unconfirmed PR in dedifferentiated chondrosarcoma. Twelve-week PFS was 73% (LPS), 44% (LMS), 36% (US), 60% (MPNST), and 38% (Other). Grade 3-4 adverse events: oral mucositis (12%), anemia (14%), platelet count decreased (14%), leukopenia (22%), and neutropenia (42%). CONCLUSIONS: Alisertib was well tolerated. Occasional responses, yet prolonged stable disease, were observed. Although failing to meet the primary RR end point, PFS was promising. TRIAL REGISTRATION ID: NCT01653028.

High-risk features in radiation-associated breast angiosarcomas.

BACKGROUND: Radiation-associated breast angiosarcoma (RT-AS) is an uncommon malignancy with an incidence of less than 1 % of all soft tissue sarcomas. The overall prognosis is quite dismal with high rates of recurrences and poor overall survival. There is an obvious paucity of data regarding clinical outcomes of patients with breast RT-AS. METHODS: We identified all patients with RT-AS treated at the Memorial Sloan-Kettering Cancer Center between 1982-2011 and collected their correlative clinical information. RESULTS: We identified 79 women with RT-AS with a median age of 68 (range 36-87). The median interval between radiation and development of RT-AS was 7 years (range 3-19). The median time to local and distant recurrence was 1.29 years (95 % CI 0.72-NA) and 2.48 years (95 % CI 1.29-NA), respectively. The median disease-specific survival was 2.97 years (95 % CI 2.21-NA). Independent predictors of worse disease-specific survival included age 68 years (HR 3.11, 95 % CI 1.20-8.08, P=0.020) and deep tumors (HR 3.23, 95 % CI 1.02-10.21, P=0.046.) CONCLUSION: RT-AS has high local/distant recurrence rates, limited duration on standard chemotherapy and poor disease-specific survival.

Phase II study of the HSP90-inhibitor BIIB021 in gastrointestinal stromal tumors.

BACKGROUND: HSP90 inhibition leads to proteosomal degradation of activated KIT and has in vitro activity against gastrointestinal stromal tumors (GIST). BIIB021 is an oral non-ansamycin HSP90 inhibitor. We carried out a phase II study of BIIB021 in patients with GIST refractory to imatinib and sunitinib. PATIENTS AND METHODS: The primary end-point was metabolic partial response (mPR) as assessed by fluorodeoxyglucose positron emission tomography (FDG-PET). The secondary end-points were pharmacokinetic assessments of BIIB021 and pharmacodynamic assessments of HSP70. Twenty-three patients were treated on two schedules: 12 pts received 600 mg twice a week (BIW) and 11 patients received 400 mg three times a week (TIW). All had prior imatinib and sunitinib but stopped>14 days before starting BIIB021. RESULTS: The median age was 59 years (33-88 years), 61% male, 44% Eastern Cooperative Oncology Group 1 (ECOG1). The best response was PR by FDG-PET for five patients (3/12 at 600 mg BIW and 2/9 at 400 TIW) for an overall response rate of 22%. The response duration was 25-138 days. Adverse events (AEs) were mild to moderate. The mean Cmax was 1.5 µmol and the mean AUC was 2.9 µmol h. Cmax>1.5 µmol was associated with a decrease in standardized uptake value (SUVmax). HSP70 increased substantially following treatment. CONCLUSIONS: This study met its primary end-point. BIIB021 leads to objective responses in refractory GIST patients. Pharmacodynamic studies confirmed HSP90 inhibition. Further evaluation of BIIB021 in GIST is warranted.

Advanced well-differentiated/dedifferentiated liposarcomas: role of chemotherapy and survival.

BACKGROUND: Data regarding the role of systemic therapy in patients with advanced well-differentiated/dedifferentiated liposarcomas (WDLPS/DDLPS) are limited. METHODS: From 2000 to 2010, 208 patients with advanced WDLPS/DDLPS received chemotherapy in 11 participating institutions. Clinical and pathological data were collected by reviewing medical records. RESULTS: Median age was 63 years (range 32-84). Combination chemotherapy was delivered in 85 cases (41%) and single agent in 123 cases (59%), respectively. One hundred and seventy-one patients (82%) received an anthracycline-containing regimen. Using RECIST, objective response was observed in 21 patients (12%), all treated with anthracyclines. Median progression-free survival (PFS) was 4.6 months [95% confidence interval (CI) 3.3-5.9]. On multivariate analysis, age and performance status (PS) were the sole factors significantly associated with poor PFS. Median overall survival (OS) was 15.2 months (95% CI 11.8 -18.7). On multivariate analysis, grade and PS were the sole factors significantly associated with OS. CONCLUSIONS: Chemotherapy was associated with clinical benefit in 46% of patients with advanced WDLPS/DDLPS. OS remains poor, even though visceral metastatic disease is less frequent than in other sarcomas.

Phase I study of PD 0332991, a cyclin-dependent kinase inhibitor, administered in 3-week cycles (Schedule 2/1).

BACKGROUND: This phase I, open-label, first-in-human study determined dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of PD 0332991, an oral cyclin-dependent kinase 4/6 inhibitor with potent anti-proliferative activity in vitro/vivo. METHODS: A total of 33 patients with retinoblastoma protein-positive advanced solid tumours or non-Hodgkin's lymphoma refractory to standard therapy or for which no therapy was available received PD 0332991 once daily (QD) for 14 days followed by 7 days off treatment (21-day cycles; Schedule 2/1). RESULTS: Six patients had DLTs (18%; four receiving 200 mg QD; two receiving 225 mg QD); the MTD was 200 mg QD. Treatment-related, non-haematological adverse events occurred in 29 patients (88%) during cycle 1 and 27 patients (82%) thereafter. Adverse events were generally mild-moderate. Of 31 evaluable patients, one with testicular cancer achieved a partial response; nine had stable disease (≥10 cycles in three cases). PD 0332991 was slowly absorbed (mean T(max) 4.2 h) and eliminated (mean half-life 26.7 h). Volume of distribution was large (mean 3241 l) with dose-proportional exposure. Using a maximum effective concentration model, neutropenia was proportional to exposure. CONCLUSION: PD 0332991 was generally well tolerated, with DLTs related mainly to myelosuppression. The MTD, 200 mg QD, is recommended for phase II study.

Safety, tolerability, pharmacokinetics and pharmacodynamics of the oral cyclin-dependent kinase inhibitor AZD5438 when administered at intermittent and continuous dosing schedules in patients with advanced solid tumours.

BACKGROUND: AZD5438 is an orally bioavailable inhibitor of cyclin E-cdk2, cyclin A-cdk2 and cyclin B-cdk1 complexes. Three phase I studies assessed the clinical safety, tolerability, pharmacokinetics and pharmacodynamics of AZD5438 when administered in different dosing schedules. PATIENTS AND METHODS: AZD5438 was administered four times daily, once every 7 days (study 1), for 14 consecutive days followed by 7 days of rest (study 2), or continuously (study 3), to patients with advanced solid tumours. Dose escalation proceeded until the emergence of dose-limiting toxic effects. RESULTS: Sixty-four patients were included across the three studies (19, 17 and 28, respectively). Nausea and vomiting were the most common adverse events. When dosed continuously, 40 mg four times daily was considered intolerable, and due to safety issues, all studies were terminated prematurely. Consequently, no intolerable dose was identified during the weekly schedule. Pharmacokinetics demonstrated dose-proportional exposure, high interpatient variability and accumulation after multiple doses. Skin biopsies indicated reduced retinoblastoma protein phosphorylation at cdk2 phospho-sites; other pharmacodynamic assessments did not reveal consistent trends. CONCLUSIONS: AZD5438 was generally well tolerated in a weekly dosing schedule, but not in continuous schedules. The clinical development programme for AZD5438 was discontinued owing to tolerability and exposure data from these studies.

Development of cell-cycle inhibitors for cancer therapy.

The cell cycle governs the transition from quiescence through cell growth to proliferation. The key parts of the cell cycle machinery are the cyclin-dependent kinases (CDKS) and the regulatory proteins called cyclins. The CDKS are rational targets for cancer therapy because their expression in cancer cells is often aberrant and their inhibition can induce cell death. Inhibitors of CDKS can also block transcription.Several drugs targeting the cell cycle have entered clinical trials. These agents include flavopiridol, indisulam, AZD5438, SNS-032, bryostatin-1, seliciclib, PD 0332991, and SCH 727965. Phase i studies have demonstrated that these drugs can generally be administered safely. Phase ii studies have shown little single-agent activity in solid tumors, but combination studies with cytotoxic chemotherapy have been more promising. In hematologic malignancies, reports have shown encouraging single-agent and combination activity. Pharmacodynamic studies show that the dose and schedule of these drugs are crucial to permit maximum therapeutic effect.

Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1.

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, small-molecule antagonists of MDM2, the Nutlins, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. However, half of human cancers have mutated p53 and they are resistant to Nutlin treatment. Here, we report that treatment of the p53-mutant malignant peripheral nerve sheath (MPNST) and p53-null HCT116 cells with cisplatin (Cis) and Nutlin-3a induced a degree of apoptosis that was significantly greater than either drug alone. Nutlin-3a also increased the cytotoxicity of both carboplatin and doxorubicin in a series of p53-mutant human tumor cell lines. In the human dedifferentiated liposarcoma cell line (LS141) and the p53 wild-type HCT116 cells, Nutlin-3a induced downregulation of E2F1 and this effect appeared to be proteasome dependent. In contrast, in MPNST and HCTp53-/- cells, Nutlin-3a inhibited the binding of E2F1 to MDM2 and induced transcriptional activation of free E2F1 in the presence of Cis-induced DNA damage. Downregulation of E2F1 by small interfering RNA significantly decreased the level of apoptosis induced by Cis and Nutlin-3a treatment. Moreover, expression of a dominant-negative form of E2F1 rescued cells from apoptosis, whereas cells overexpressing wild-type E2F1 showed an increase in cell death. This correlated with the induction of the proapoptotic proteins p73alpha and Noxa, which are both regulated by E2F1. These results indicate that antagonism of MDM2 by Nutlin-3a in cells with mutant p53 enhances chemosensitivity in an E2F1-dependent manner. Nutlin-3a therefore may provide a therapeutic benefit in tumors with mutant p53 provided it is combined with chemotherapy.

A phase I study of erlotinib in combination with gemcitabine and radiation in locally advanced, non-operable pancreatic adenocarcinoma.

PURPOSE: To determine the maximum tolerated dose (MTD) of erlotinib when administered concurrently with twice weekly gemcitabine and radiation therapy (RT) for locally advanced pancreatic cancer, assess the safety and toxicity profile of this combination and secondarily evaluate response, time to tumor progression and overall survival. METHODS: Patients with untreated locally advanced pancreas cancer were treated with daily erlotinib in combination with gemcitabine 40 mg/m(2)/30 min twice weekly and RT delivered at 180 cGy/day in 28 fractions over 5.5 weeks for a total of 5040 cGy. Erlotinib was dose escalated in successive cohorts (100 mg, 125 mg). When the MTD was determined, the cohort was expanded to better define toxicity and preliminarily efficacy. All patients were surgically staged. After chemoradiation, patients received maintenance weekly gemcitabine 1000 mg/m(2) on days 1 and 8 of a 21 day cycle and daily erlotinib for four cycles. RESULTS: Three patients were treated at dose level 1 (erlotinib 100 mg) without limiting toxicity. Two of six patients at dose level 2 (erlotinib 125 mg) had dose-limiting toxicities, neutropenia and thrombocytopenia, causing dose delay and elevated liver enzymes. The MTD for erlotinib in combination with twice weekly gemcitabine-based chemoradiation was 100 mg/day. Eleven additional patients were treated at dose level 1. All twenty patients were assessable for toxicity. Seventeen patients were assessable for response. The partial response rate was 35% and 53% had stable disease. The median survival for all patients was 18.7 months. CONCLUSION: In combination with fixed dose gemcitabine at 40 mg/m(2) twice weekly and radiation at 180 cGy/day, the MTD of erlotinib was found to be 100 mg/day. This is a relatively well tolerated, biologically active combination in a poor prognostic cancer.

Phase I dose-finding study of weekly docetaxel followed by flavopiridol for patients with advanced solid tumors.

PURPOSE: Flavopiridol is a cyclin-dependent kinase inhibitor that enhances docetaxel-induced apoptosis in a sequence-specific manner. In vivo, docetaxel must precede flavopiridol by at least 4 h to induce this effect. We conducted a phase I trial of weekly, sequential docetaxel followed 4 h later by flavopiridol in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Docetaxel at a fixed dose of 35 mg/m2 was administered over 30 min, followed 4 h later by escalating doses of flavopiridol, ranging from 20 to 80 mg/m2 in successive cohorts, administered weekly over 1 h. This schedule was repeated for 3 weeks of each 4-week cycle. RESULTS: Twenty-seven evaluable patients were enrolled. The combination was well tolerated, with one dose-limiting toxicity occurring at flavopiridol 70 mg/m2 (grade 3 mucositis) and one dose-limiting toxicity at 80 mg/m2 (grade 4 neutropenia). We observed 1 complete response in a patient with pancreatic carcinoma and 4 partial responses in pancreatic (1), breast (2), and ovarian (1) cancer patients. Stable disease was seen in 10 patients. Pharmacokinetic studies showed Cmax ranging from 1.49 +/- 0.69 micromol/L (flavopiridol 20 mg/m2) to 4.54 +/- 0.08 micromol/L (flavopiridol 60 mg/m2) in cycle 1. CONCLUSIONS: Treatment with weekly, sequential docetaxel followed by flavopiridol is an effective and safe regimen at all flavopiridol dose levels. The pharmacokinetic data indicate that concentrations of flavopiridol that enhance the effects of docetaxel both in vitro and in vivo can be achieved. Clinical activity is encouraging, even in patients who have received a prior taxane and in patients with gemcitabine-refractory metastatic pancreatic cancer.

Obstruction of BRAFV600E transcription by complementary PNA oligomers as a means to inhibit BRAF-mutant melanoma growth.

Peptide nucleic acid (PNA) oligomers are DNA mimics, which are capable of binding gene sequences 1000-fold more avidly than complementary native DNA by strand invasion and effectively obstruct transcription. Irreversibly obstructing the transcription or replication of a gene sequence, such as BRAFV600E, offers a potential route to specifically target the cancer cell itself. We have employed PNA oligomers to target BRAFV600E in a sequence-specific complementary manner. These PNAs have been modified by appending configurationally stabilizing cationic peptides in order to improve their cellular delivery and target avidity. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAFV600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAFV600E-mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show increased tumor growth delay and necrosis with the BRAFV600E-complementary PNA-peptide conjugates as compared with the saline and scrambled PNA sequence controls. Similarly, quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling expression with this approach. PNA-delivery peptide conjugates represent a novel way to target BRAFV600E and represent a new approach in targeting selective oncogenes that induce tumor growth.

Protein kinase C: a novel target for inhibiting gastric cancer cell invasion.

BACKGROUND: Gastric adenocarcinoma is a common neoplasm worldwide. Patients with completely resected disease often have locoregional recurrence, and adjuvant chemotherapy has failed to reduce the common occurrence of metastases. Protein kinase C (PKC) is thought to be important in tumor cell invasion, but its relationship to gastric cancer cell invasion, and thus metastases, remains unexplored. We recently identified and established invasive and noninvasive human gastric adenocarcinoma cell lines, which can now be used to test agents for inhibition of tumor cell invasion by inhibition of PKC activity. PURPOSE: The objectives were (a) to test threo-dihydrosphingosine (SPC100221), a specific inhibitor of PKC at its regulatory site, and staurosporine, a potent but nonspecific inhibitor of PKC at its catalytic site, for their effects on gastric cancer cell invasion in vitro and (b) to determine whether the expression of PKC isoforms can distinguish invasive from noninvasive gastric cancer cells. METHODS: Gastric cancer cell invasion through Matrigel-coated Nuclepore filters in the Boyden chamber assay was analyzed in the presence of graded concentrations of SPC100221 and staurosporine. The invasive SK-GT-1 and SK-GT-5 cell lines and the noninvasive SK-GT-2 and SK-GT-4 cell lines were used. PKC isoform expression was determined by reverse transcription of messenger RNAs to complementary DNA and subsequent amplification by the polymerase chain reaction. RESULTS: The effects of staurosporine and SPC100221 on tumor cell invasion were tested at drug concentrations that did not inhibit cell proliferation, as evidenced by [3H]thymidine uptake. Staurosporine and SPC100221 at subtoxic doses inhibited human gastric cancer cell invasion by 50% at 5 x 10(-9) M and 2 x 10(-7) M, respectively. The expression of PKC beta was observed in the invasive but not the noninvasive gastric cancer cells. Both types of cells, however, expressed the PKC alpha and PKC gamma isoforms. CONCLUSIONS: Gastric cancer cell invasion can be inhibited by PKC inhibitors, and expression of PKC beta may be a marker of invasiveness in gastric cancer. IMPLICATIONS: PKC appears to represent a new target for inhibition of gastric cancer cell invasion, and SPC100221, in view of its PKC specificity, may provide a model for future drug development in this area. Moreover, PKC beta may have a fundamental role in the development of invasive potential in gastric cancer.

Inhibition of invasion of invasive human bladder carcinoma cells by protein kinase C inhibitor staurosporine.

To study the effect of the protein kinase C (PKC) inhibitor staurosporine on invasion, we selected the invasive human bladder carcinoma cell line EJ. Total PKC activity was more than twofold higher in the EJ cells than in RT4 cells (superficial human bladder carcinoma cells), which do not pass through an artificial basement membrane. There was more PKC activity in the cytosol than in the membrane of EJ cells. Staurosporine, at nontoxic concentrations, inhibited the invasion of EJ cells through an artificial basement membrane in a dose-dependent manner. Staurosporine caused a dose-dependent inhibition of cell motility but did not inhibit cell attachment. Staurosporine represents a new agent for the inhibition of tumor cell invasion and may prove useful in studying the mechanisms responsible for this phenomenon.

Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells.

BACKGROUND: Protein kinase C (PKC) is a family of enzymes that function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis. PURPOSE: We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or their drug-resistance status. METHODS: SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 microM) alone, MMC (5 micrograms/mL) alone, or a combination of safingol (50 microM) and MMC (5 micrograms/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells. RESULTS: As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- SD) of the cells, and MMC alone increased that level to 18% +/- 1%. However, the combination of safingol and MMC induced apoptosis in 39% +/- 1% of the cells (P < .001, for the drug combination versus MMC alone). With MKN-74 cells, safingol alone induced apoptosis in 8% +/- 3% of the cells, whereas MMC alone induced apoptosis in 40% +/- 4% of treated cells and the combination of safingol and MMC induced apoptosis in 83% +/- 4% of the cells. Similar results were obtained with the TdT assay. Simultaneous exposure of cells to safingol and PMA abrogated the safingol-mediated enhancement of MMC-induced apoptosis. CONCLUSIONS: The PKC inhibitor safingol enhances the cytotoxic effect of the chemotherapeutic agent MMC in gastric cancer cells by promoting drug-induced apoptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.

Protein kinase C activity and multidrug resistance in MOLT-3 human lymphoblastic leukemia cells resistant to trimetrexate.

It has been suggested that protein kinase C (PKC) plays a role in multidrug resistance (MDR). In this study we assayed PKC activity in MOLT-3 human acute lymphoblastic leukemia cells and found an approximately 50% decrease in activity in MDR sublines made resistant to the lipophilic antifolate trimetrexate, when compared with trimetrexate-sensitive parent cells. The PKC activity of a methotrexate-resistant subline without MDR (MOLT-3/MTX10,000) was identical to that of parent cells. Although a downward trend was noted in PKC activity in the membrane fraction of cells with increasing trimetrexate resistance, there was no absolute correlation between the degree of MDR and the relative decrease in PKC activity. Using the same method, we also confirmed an over 6-fold increase in PKC activity in the MDR human breast cancer subline MCF-7/DOXR when compared with the sensitive parent cell line, MCF-7/WT. Because of this divergent relationship between relative PKC activity and MDR, we tested the effect of PKC inhibition and activation on drug resistance. The PKC inhibitor staurosporine, at both subtoxic and toxic concentrations as well as at concentrations shown to be inhibitory to PKC, failed to increase drug resistance of parent and resistant MOLT-3 cells and decrease drug resistance of MCF-7/WT and MCF-7/DOXR cells. Short-term exposure to 3-phorbol-12-myristate-13-acetate, which activated PKC 7.0-fold and 4.7-fold, respectively, in the membrane of MOLT-3 and resistant cells, resulted in small (1.3- to 1.8-fold), approximately equivalent, increases (rather than decreases) in resistance to doxorubicin, whereas for vincristine no consistent trend was observed. Identical results were also obtained with phorbol-12,13-dibutyrate. These results indicate that PKC activity can be decreased and increased in MDR cells. Both staurosporine inhibition and phorbol ester activation failed to produce changes in drug resistance that would be considered consistent with the resulting degree of PKC activity. Short-term phorbol ester exposure can change the sensitivity of the cells to doxorubicin without changing the relative drug resistance. PKC activity in these cells may then be unrelated to MDR.

High frequency of simultaneous loss of p16 and p16beta gene expression in squamous cell carcinoma of the esophagus but not in adenocarcinoma of the esophagus or stomach.

Quantitative reverse transcription PCR (RT-PCR) was used to measure gene expressions (relative mRNA levels) of p16 and the alternate transcript pl6beta in esophageal and gastric tumors. p16 gene expression was undetectable in 13 of 25 esophageal squamous cell carcinomas. In 11 of these tumors, pl6beta was simultaneously missing whereas two of the pl6-deficient tumors still expressed p16beta. Among 34 esophageal adenocarcinomas and 11 gastric adenocarcinomas, only one tumor lacked p16 expression and all tumors expressed p16beta. p16 sequences were not detectable by PCR in genomic DNA from tumors lacking both p16 and p16beta mRNA, suggesting that the simultaneous loss of both gene expressions resulted from homozygous genomic deletion of the p16 gene. However, DNA from tumors that lacked p16 mRNA but expressed pl6beta did contain the p16 gene, consistent with loss of p16 expression in these tumors by transcriptional suppression. No point mutations in p16 cDNA were detected among 12 that were sequenced, but one p16 cDNA from a squamous cell carcinoma had a 19-base deletion, possibly indicating a splice-site mutation. Among those tumors that expressed p16 mRNA, the gene expression values of both p16 and pl6beta varied over a wide range. In some cases, p16 expression was detectable but low, suggesting that down-regulation of p16 expression may be used in some cases to achieve the funtional equivalent of gene deletion or transcriptional silencing. These results demonstrate that p16 expression patterns differ based on tumor histology and origin. Homozygous deletion of p16 appears to be common in esophageal squamous cell carcinomas but in adenocarcinomas, both gene deletion and transcriptional silencing of p16 were infrequent.

Phase I trial of dose-intense liposome-encapsulated doxorubicin in patients with advanced sarcoma.

PURPOSE: To define the maximum-tolerated dose (MTD) of liposome-encapsulated doxorubicin (LED) when used every 2 weeks with granulocyte colony-stimulating factor (G-CSF) in patients with advanced soft tissue sarcoma. PATIENTS AND METHODS: Doxorubicin encapsulated in egg phosphatidylcholine/cholesterol liposomes was given to patients with sarcoma in a disease-specific phase I trial. The initial dose was 75 mg/m2 with G-CSF 5 micrograms/kg. The MTD was defined as the highest dose that could be given every 2 weeks. RESULTS: Twenty-nine patients participated in this study. Major toxicities included myelosuppression, nausea and vomiting, fatigue, and mucositis. Eight patients were hospitalized for nadir fever. No cardiotoxicity was seen. The MTD was LED 105 mg/m2 with G-CSF 5 micrograms/kg. LED 120 mg/m2 resulted in tolerable, albeit prominent, acute toxicity, but did not permit recycling of therapy on day 15. Among 26 patients with soft tissue sarcoma, 23 had measurable disease, of whom three achieved a partial response (13%; 95% confidence interval, 2% to 34%). CONCLUSION: LED can be administered every 2 weeks at a dose of 105 mg/m2 with G-CSF support, which provides a dose-intensity of 52.5 mg/m2/wk. To exceed this intensity, the dose of LED that would have to be administered every 3 weeks would be greater than 157.5 mg/m2. A formal phase II trial is needed to estimate better the true response rate.

Phase II study of the cyclin-dependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma.

PURPOSE: Flavopiridol is the first cyclin-dependent kinase inhibitor to enter clinical trials. Activity in gastric cancer xenografts and in a patient with gastric cancer on the phase I trial led to this phase II study of flavopiridol in patients with metastatic gastric cancer. PATIENTS AND METHODS: Sixteen patients were entered onto the study, and 14 were assessable for response. Flavopiridol was administered initially at a dose of 50 mg/m(2)/d by continuous infusion for 72 hours every 2 weeks. Assessment of plasma pharmacokinetics was performed in all patients. Peripheral mononuclear cells were collected throughout the 72-hour infusion for determinants of apoptosis. RESULTS: There were no major objective responses (exact confidence interval 0% to 23%). One patient achieved a minor response in his liver metastases, though the primary progressed. Other patients exhibited histologic and radiographic evidence of tumor necrosis. Common toxicities included fatigue in 93% of patients (grade 3 or 4 in 27%) and diarrhea in 73% of patients (grade 3 or 4 in 20%). Five patients (33%) developed venous thromboses at the central catheter tip. The studies performed on peripheral mononuclear cells indicated no induction of apoptosis. CONCLUSION: Flavopiridol administered as a single agent for 72 hours every 14 days is inactive in the treatment of gastric cancer. The drug also induced an unexpected higher incidence of vascular thrombosis and fatigue than was anticipated from the phase I trials. Future development of flavopiridol will depend on other doses and schedules in combination with chemotherapy.

Phase I clinical and pharmacologic study of weekly cisplatin combined with weekly irinotecan in patients with advanced solid tumors.

PURPOSE: In vitro synergy between cisplatin and irinotecan (CPT-11) has been reported. We designed a combination schedule of these agents to maximize the potential for synergistic interaction. PATIENTS AND METHODS: To maximize the opportunity for synergy, we divided the cisplatin into four consecutive weekly treatments, followed by a 2-week rest. Each dose of cisplatin was immediately followed by a dose of irinotecan. The dose of cisplatin was fixed at 30 mg/m2/wk. The initial irinotecan dose was 50 mg/m2/wk and this was escalated by 30% increments in successive cohorts of three to six patients to establish the maximum-tolerated dose (MTD). Pharmacokinetics of irinotecan and its metabolites, SN-38 and SN-38 glucuronide (SN-38G), were analyzed. RESULTS: Of 35 patients with solid tumors enrolled onto this trial, 30 were assessable for toxicity and response. The MTD for this regimen was 30 mg/m2/wk of cisplatin plus 50 mg/m2/wk of irinotecan in previously treated patients and 30 mg/m2/wk of cisplatin plus 65 mg/m2/wk of irinotecan in chemotherapy-naive patients. Neutropenia was the dose-limiting toxicity (DLT) encountered in this trial. Diarrhea was infrequent and rarely dose-limiting. Seven of 30 assessable patients achieved a partial response. No alteration in irinotecan, SN-38, or SN-38G pharmacokinetics resulted from the administration of cisplatin with irinotecan. CONCLUSION: The administration of cisplatin and irinotecan on this weekly schedule provides a practical and well-tolerated regimen that has the potential to maximize any clinical synergy between the two agents. Evidence of substantial clinical activity was seen in this phase I study.