Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma.

Romidepsin (depsipeptide or FK228) is a histone deacetylase inhibitor, one of a new class of agents active in T-cell lymphoma. A phase 2 trial was conducted in cutaneous (CTCL) and peripheral (PTCL) T-cell lymphoma. Major and durable responses in CTCL supported the approval of romidepsin for CTCL. Forty-seven patients with PTCL of various subtypes including PTCL NOS, angioimmunoblastic, ALK-negative anaplastic large cell lymphoma, and enteropathy-associated T-cell lymphoma were enrolled. All patients had received prior therapy with a median of 3 previous treatments (range 1-11); 18 (38%) had undergone stem-cell transplant. All patients were evaluated for toxicity; 2 patients discovered to be ineligible were excluded from response assessment. Common toxicities were nausea, fatigue, and transient thrombocytopenia and granulocytopenia. Complete responses were observed in 8 and partial responses in 9 of 45 patients, for an overall response rate of 38% (95% confidence interval 24%-53%). The median duration of overall response was 8.9 months (range 2-74). Responses were observed in various subtypes, with 6 responses among the 18 patients with prior stem-cell transplant. The histone deacetylase inhibitor romidepsin has single agent clinical activity associated with durable responses in patients with relapsed PTCL.

Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver.

Preneoplastic and neoplastic liver nodules and hepatocytes isolated from regenerating rat liver have been shown to be resistant to a broad range of carcinogenic agents. This phenomenon was studied by measuring the expression of the multidrug-resistant (mdr) gene in normal liver cells and in preneoplastic and neoplastic nodules and regenerating liver. Levels of messenger RNA for the mdr gene, which encodes P-glycoprotein, were elevated in both preneoplastic and neoplastic lesions. Expression of the mdr gene also reached high levels in regenerating rat liver 24 to 72 hours after partial hepatectomy. These results show that the expression of the mdr gene can be regulated in liver and is likely to be responsible for part of the multidrug-resistance phenotype of carcinogen-initiated hepatocytes and regenerating liver cells.

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.

Various methods of analysis of mdr-1/P-glycoprotein in human colon cancer cell lines.

BACKGROUND: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels. PURPOSE: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists. METHODS: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure. RESULTS: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil. CONCLUSIONS: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative. IMPLICATIONS: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo.

Expression of a multidrug resistance gene in human cancers.

Many cancers have been cured by chemotherapeutic agents. However, other cancers are intrinsically drug resistant, and some acquire resistance following chemotherapy. Cloning of the cDNA for the human MDR1 gene (also known as PGY1), which encodes the multidrug efflux protein P-glycoprotein, has made it possible to measure levels of MDR1 RNA in human cancers. We report the levels of MDR1 RNA in greater than 400 human cancers. MDR1 RNA levels were usually elevated in untreated, intrinsically drug-resistant tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas, as well as in carcinoid tumors, chronic myelogenous leukemia in blast crisis, and cell lines of non-small cell carcinoma of the lung (NSCLC) with neuroendocrine properties. MDR1 RNA levels were occasionally elevated in other untreated cancers, including neuroblastoma, acute lymphocytic leukemia (ALL) in adults, acute nonlymphocytic leukemia (ANLL) in adults, and indolent non-Hodgkin's lymphoma. MDR1 RNA levels were also increased in some cancers at relapse after chemotherapy, including ALL, ANLL, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma. Many types of drug-sensitive and drug-resistant tumors, including NSCLC and melanoma, contained undetectable or low levels of MDR1 RNA. The consistent association of MDR1 expression with several intrinsically resistant cancers and the increased expression of the MDR1 gene in certain cancers with acquired drug resistance indicate that the MDR1 gene contributes to multidrug resistance in many human cancers. Thus, evaluation of MDR1 gene expression may prove to be a valuable tool in the identification of individuals whose cancers are resistant to specific agents. The information may be useful in designing or altering chemotherapeutic protocols in these patients.

Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines.

Human KB cells with increasing resistance to colchicine and other chemotherapeutic agents have been isolated in four sequential steps. This report describes the characterization of drug uptake in the parent and four mutant cell lines. Drug uptake in these cell lines occurred via a nonsaturable process. In general, drug accumulation decreased with increasing drug resistance; this relationship was seen best with colchicine, vincristine, vinblastine, and daunomycin and, to a lesser extent, with actinomycin D. The accumulation of dexamethasone, an agent to which all lines were equally sensitive, was similar for the parent and the four mutants. Drug efflux occurred rapidly, and differences among the various cell lines could be detected within the first minute. In the more resistant lines, a greater percentage of the drug was released more rapidly, although the absolute amount of drug released was less. Verapamil partially reversed the multiple drug-resistance phenotype by increasing the initial rate of uptake and accumulation of drugs in the resistant cell lines without an apparent effect on drug efflux. The results suggest that, in this human epithelial cell, the development of resistance to multiple drugs is complex, with changes in drug uptake, accumulation, and efflux.

Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum (II) in human ovarian cancer cell lines.

A human ovarian cancer cell line, A2780, derived from an untreated ovarian cancer patient and relatively sensitive to cisplatin was treated by stepwise incubation with cisplatin to produce a cisplatin-resistant variant, 2780CP. The relative abilities of these cell lines to repair cisplatin-induced damage to cellular DNA then was examined by measure of [3H]thymidine incorporation into normal density DNA separated from bromodeoxyuridine-substituted DNA on alkaline cesium chloride gradients. These studies revealed that primary cisplatin resistance present in 2780CP was associated with a near twofold-increased ability to repair damage induced by the drug under conditions where 2780CP was approximately 5-fold resistant to cisplatin. Aphidicolin, a specific inhibitor of DNA polymerase alpha, showed a dose-dependent capacity to inhibit DNA repair in this system with maximum inhibition of 63% at 4 micrograms/ml. It was also found that inhibition of DNA repair during and shortly after cisplatin exposure resulted in an approximately threefold increase in the cytotoxicity of cisplatin as monitored by clonogenic cell survival in the resistant but not the sensitive parental cell line.

Acquisition of hormone-independent growth in MCF-7 cells is accompanied by increased expression of estrogen-regulated genes but without detectable DNA amplifications.

A hormone-independent but hormone-responsive subpopulation (MCF7/MIII) of the hormone-dependent MCF-7 human breast cancer cell line (R. Clarke et al., Proc. Natl. Acad. Sci. USA 86: 3649-3653, 1989) was further passaged in ovariectomized nude mice and re-established in vitro as the continuous cell line MCF7/LCC1. The lag time to the appearance of proliferating tumors in ovariectomized animals is significantly reduced in MCF7/LCC1 when compared with MCF7/MIII cells. In gel denaturation/renaturation analysis of tumor, genomic DNA does not reveal significant differences in the pattern of detectable DNA amplifications between parent MCF-7 cells and MCF7/LCC1 cells. In the absence of estrogen, steady-state levels of phosphoinositol turnover are similar in both MCF-7 and MCF7/LCC1 cells, but turnover is increased by estrogen only in MCF-7 cells. MCF7/MIII and MCF7/LCC1, but not MCF-7 cells, express a high baseline level of the estrogen-regulated pS2 mRNA. The baseline level of expression of progesterone receptor protein, but not mRNA, is higher in MCF7/LCC1 when compared with either MCF-7 or early passage MCF7/MIII cells. However, while the estrogen receptor is also an estrogen-regulated gene, MCF7/MIII and MCF7/LCC1 cells retain estrogen receptor levels equivalent to the parental MCF-7 cells. These data indicate that progression to hormone independence can occur without major gene amplifications or a high constitutive induction of phosphoinositide metabolism. Thus, DNA amplifications may be acquired during the early initiation and/or promotional events of carcinogenesis. Significantly, acquisition of a hormone-independent but responsive phenotype in human breast cancer is associated with perturbations in the expression of specific estrogen-regulated genes.

Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance.

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.

Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene.

The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors. We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA. Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro. These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.

EPOCH chemotherapy: toxicity and efficacy in relapsed and refractory non-Hodgkin's lymphoma.

PURPOSE: Based on in vitro evidence that tumor cells are less resistant to prolonged exposure to low concentrations of the natural product class, compared with brief higher concentration exposure, we developed a chemotherapy regimen (etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone [EPOCH]) in which the natural products are administered as a continuous infusion. PATIENTS AND METHODS: This is a phase II study of etoposide, vincristine, and doxorubicin, administered as a 96-hour continuous infusion, with intravenous (IV) bolus cyclophosphamide and oral prednisone (EPOCH) in 74 consecutive patients who relapsed from or failed to respond to most of the same drugs administered on a bolus schedule. Patients with aggressive lymphomas who achieved a good response after EPOCH were eligible to undergo bone marrow transplantation. RESULTS: Patients with intermediate- or high-grade lymphoma comprised 76% of this series and 77% had stage IV disease. Seventy-one percent had previously received all of the drugs contained in the EPOCH regimen and 92% had received at least four of the drugs. Seventy patients were assessable for response, of whom 19 (27%) achieved a complete remission (CR) and 42 (60%) a partial remission (PR). Among 21 patients who had no response to prior chemotherapy, 15 (71%) responded, but only one achieved a CR. Patients who relapsed from an initial CR had a 100% response rate, with 76% CRs. With a median potential follow-up duration of 19 months, there was a 28% probability of being event-free at 1 year. Toxicity was primarily hematologic with neutropenia during 51% of cycles, but only a 17% incidence of febrile neutropenia. Gastrointestinal, neurologic, and cardiac toxicity were minimal. CONCLUSION: EPOCH chemotherapy was well tolerated and highly effective in patients who were resistant to or relapsed from the same drugs administered on a bolus schedule, suggesting that continuous infusion of the natural drug component of this regimen is capable of partially reversing drug resistance and reducing toxicity. Dose-intensity (DI) was > or = that achieved in primary treatment regimens for aggressive lymphomas.

Paclitaxel in doxorubicin-refractory or mitoxantrone-refractory breast cancer: a phase I/II trial of 96-hour infusion.

PURPOSE: A phase I study of paclitaxel infused over 96-hours was performed to determine toxicity, maximum-tolerated dose (MTD), and pharmacokinetics in patients with incurable lymphomas and solid tumors. A phase II study was performed at the MTD of paclitaxel in patients with doxorubicin/mitoxantrone-refractory metastatic breast cancer. PATIENTS AND METHODS: In the phase I study, paclitaxel dose levels ranged from 120 to 160 mg/m2, administered on a 21-day cycle. Patients with metastatic breast cancer who had either no response or a partial response (PR) to doxorubicin or mitoxantrone and had measurable disease were eligible for the phase I and II studies. Expression of the multidrug resistance (mdr-1) gene was determined in tumor biopsies by mRNA quantitative polymerase chain reaction. RESULTS: Twelve patients received a total of 73 cycles of paclitaxel on the phase I study. Dose-limiting mucositis and/or grade IV granulocytopenia was reached at 160 mg/m2, and 140 mg/m2 was selected as the phase II dose. Thirty-six consecutive patients with metastatic breast cancer were treated, of whom three were not assessable. The median age was 49 years, with disease in the liver and/or lung in 76%. Patients received a median of two prior regimens for metastatic disease, and 73% had no response to prior doxorubicin or mitoxantrone. Of 33 patients treated with paclitaxel, 16 patients (48%) achieved a PR and five (15%) achieved a minor response (MR). With a median potential follow-up duration of 60 weeks, the median progression-free and overall survival durations were 27 and 43 weeks, respectively. No correlation was found between extent of prior treatment or prior response to doxorubicin/mitoxantrone, and response to paclitaxel. Paclitaxel pharmacokinetics showed a correlation between both granulocyte and mucosal toxicity, and serum steady-state concentrations (Css) more than 0.07 mumol/L. Patients with liver metastases had significantly decreased paclitaxel clearance and higher paclitaxel Css. Levels of mdr-1 were uniformly low in all tumor biopsies studied. CONCLUSION: The recommended phase II dose of paclitaxel is 140 mg/m2 in patients without liver metastases and 105 mg/m2 in patients with liver metastases. Ninety-six-hour infusions of paclitaxel were effective and well tolerated in patients with doxorubicin/mitoxantrone-refractory breast cancer. Prolonged infusion schedules may be more effective than shorter schedules and deserve further study.

Phase II study of paclitaxel in relapsed non-Hodgkin's lymphomas.

PURPOSE: To assess the efficacy and toxicity of paclitaxel administered as a 96-hour infusion to patients with relapsed non-Hodgkin's lymphomas (NHLs). PATIENTS AND METHODS: Eligible patients had relapsed NHL and measurable disease and were considered incurable. Paclitaxel was infused at a dose of 140 mg/m2 every 3 weeks. Premedications to prevent paclitaxel hypersensitivity reactions were not administered and no patients received corticosteroids. Expression of the multidrug resistance (mdr-1) gene was determined in tumor from 17 patients by mRNA quantitative polymerase chain reaction (PCR). RESULTS: Thirty-one patients received a total of 99 cycles of paclitaxel. Two patients were not assessable for response. The median age was 50 years, 71% had stage IV disease, and intermediate/high-grade histology was present in 65% of patients. Patients had received a median of three prior chemotherapy regimens, and 68% of patients had responded to the previous chemotherapy (chemotherapy-sensitive). Of 29 assessable patients, five (17%) achieved a partial response (PR). With a median potential follow-up time of 17 months, the median event-free and overall survival durations were 1.6 and 7.5 months, respectively. No correlation was found between response to paclitaxel and extent of prior treatment or response. The mdr-1 gene was easily detectable in 14 of 17 tumor biopsies, but was low in all but one sample. The most serious toxicity was grade 4 neutropenia, which occurred during 14% of cycles. CONCLUSION: Paclitaxel was well tolerated, but had a low response rate in patients with relapsed NHLs. There was no clear association between response to paclitaxel and extent of our response to prior treatment. Most patients had chemotherapy-sensitive disease, which suggests that the low response rate to paclitaxel was probably not due to general chemotherapy resistance. Paclitaxel provided good palliation in a minority of patients and is a reasonable agent to consider for use in patients who have failed to respond to standard chemotherapy.

Expression of the multidrug resistance, MDR1, gene in neuroblastomas.

Metastatic neuroblastoma is a childhood malignancy that is frequently responsive to chemotherapy with doxorubicin, vincristine, and teniposide (VM26), among other drugs, but in the majority of treated patients, the tumor recurs during or after chemotherapy. In this work, we have examined the hypothesis that the development of resistance to chemotherapy in neuroblastoma might be related to the expression of the human MDR1 gene, which encodes a multidrug transporter that functions as an energy-dependent drug efflux pump. RNA samples from 49 neuroblastomas were analyzed, including 31 from untreated and 18 from treated patients. MDR1 RNA was detectable in the majority of treated and untreated tumors using a sensitive, semiquantitative slot blot assay. Of the samples from treated patients, five of 18 were found to have high MDR1 RNA levels, whereas only three of 31 from untreated patients had high MDR1 levels, a statistically significant difference (P less than .01). These results show that high levels of MDR1 RNA are often associated with resistance to chemotherapy in neuroblastoma and suggest that they may contribute to this resistance. Many of the neuroblastoma samples were also evaluated for N-myc amplification but there was no correlation between N-myc copy number and the level of MDR1 mRNA expression.

Role of a doxorubicin-containing regimen in relapsed and resistant lymphomas: an 8-year follow-up study of EPOCH.

PURPOSE: Curative up-front regimens for non-Hodgkin's lymphomas contain doxorubicin, vincristine, and cyclophosphamide, whereas salvage regimens generally contain non-cross-resistant agents. We hypothesized that up-front agents may be highly effective for salvage and developed an infusional regimen based on in vitro evidence of increased efficacy. PATIENTS AND METHODS: A prospective phase II study of etoposide, vincristine, and doxorubicin over 96 hours with bolus cyclophosphamide and oral prednisone (EPOCH) was performed in 131 patients with relapsed or resistant lymphoma. RESULTS: Seventy-nine percent of patients had aggressive histologies, 46% were considered high risk by the International Prognostic Index, and 34% had resistant disease. Eighty-eight percent of patients had received at least four of the agents in EPOCH, and 94% had received doxorubicin. In 125 assessable patients, 29 (24%) achieved complete responses and 60 (50%) achieved partial responses. Among 42 patients with resistant disease, 57% responded, and in 28 patients with relapsed aggressive de novo lymphomas, 89% responded with 54% complete responses. With a median follow-up of 76 months, the overall and event-free survivals (EFS) were 17.5 and 7 months, respectively. In 33 patients with sensitive aggressive disease who did not receive stem-cell transplantation, EFS was 19% at 36 months. Toxicity was primarily hematologic, with an 18% incidence of febrile neutropenia. No clinically significant cardiac toxicity was observed, despite no maximum cumulative doxorubicin dose. CONCLUSION: EPOCH is highly effective in patients who had previously received most/all of the same drugs and produces durable remissions in curable subtypes. Salvage regimens need not contain non-cross-resistant agents, and infusional schedules may partially reverse drug resistance and reduce toxicity.

Controlled trial of dexverapamil, a modulator of multidrug resistance, in lymphomas refractory to EPOCH chemotherapy.

PURPOSE: Overexpression of the multidrug resistance gene (mdr-1) is present in up to 60% of relapsed lymphomas. To study its role in lymphomas, we conducted a controlled trial of dexverapamil, an inhibitor of the mdr-1 gene product, P-glycoprotein (Pgp), in lymphomas refractory to etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy. PATIENTS AND METHODS: Eligible patients had recurrent Hodgkin's (HD) or non-Hodgkin's lymphomas (NHL) and measurable disease. Patients initially received EPOCH alone and those with stable tumor over two cycles or progressive disease crossed over to receive dexverapamil and EPOCH on subsequent cycles. Dexverapamil was escalated eight dose levels, from 240 to 1,200 mg/m2/d. When possible, serial biopsies were obtained to measure mdr-1 expression by quantitative polymerase chain reaction (PCR). RESULTS: Of 154 patients entered onto the trial, 109 had NHL and 45 had HD. The median age was 44 years, 67% had stage IV disease, and the median number of prior regimens was two (range, one to 12) in NHL and one (range, one to four) in HD. Sixty-four patients (42%) crossed over, of which eight were not assessable. The maximum-tolerated dose of dexverapamil was 900 mg/m2/d. Among 41 NHL patients (excluding mycosis fungoides), there were three complete responses (CRs) and two partial responses (PRs) (12%) and five minor responses (MRs); two of 10 HD patients achieved PRs. The mdr-1 level was measured in 44 biopsies from 19 patients. Pretherapy, mdr-1 was low (median, 2.5 U) but increased (median, 12.2 U) at crossover. Of six patients with mdr-1 levels greater than 15 U, three responded to dexverapamil, while only one of eight patients with mdr-1 levels less than 15 U responded. EPOCH and dexverapamil were well tolerated, but compared with EPOCH alone, produced more hematologic toxicity. CONCLUSION: These results suggest that Pgp plays a role in clinical drug resistance of lymphomas. However, they also suggest that mechanisms other than Pgp are prominent in heavily pretreated patients and that, although Pgp inhibition may be necessary, it is probably insufficient. Earlier intervention with dexverapamil may be more effective and warrants further study.

Implantation of recombinant rat myocytes into adult skeletal muscle: a potential gene therapy.

The ability of skeletal muscle to regenerate provides an excellent therapeutic entry, via genetic engineering, for correcting diseases of skeletal muscle and other tissues. We have used a retrovirus to transfer the cDNA for the human multidrug transporter, encoded by the MDR1 gene, into the genomes of the rat muscle cell line L6 and into primary rat myocytes. The MDR1 gene confers drug resistance to cells, and thus serves as a selectable marker in vitro. In cultured cells, the retroviral promoter-driven human MDR1 cDNA was shown to be stable in the presence or absence of drug selection or muscle cell fusion. MDR1 mRNA was synthesized, as shown by RNA blot analysis and in situ hybridization. The protein product was localized to the plasma membrane of transduced myocytes and myotubes by immunofluorescence. As a model for skeletal muscle gene therapy, transduced L6 myocytes were implanted into the tibialis anterior muscle of Wistar rats. The retroviral sequences of the human MDR1 gene and its mRNA were present in the muscles of Wistar rats 5 days, but not 12 days, after implantation, possibly because of immunorejection. On the other hand, the human MDR1 cDNA was stable in the tibialis anterior muscle of nude mice, which are incapable of immunorejection, at least 4 weeks after implantation of myocytes. Immunosuppression of Wistar rats with cyclosporine A delayed immunorejection of recombinant myocytes, and MDR1 cDNA and mRNA was detected 3-4 weeks after implantation. In situ hybridization revealed that injected recombinant myocytes remain in discrete foci in adult rodent skeletal muscle and express MDR1 mRNA for at least 30 days in nude mice and cyclosporine-treated rats.