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S L Gerson

Publications and source records attributed to S L Gerson.

At least 19 recordsLinked to original sources
PubMed2003

Marked inactivation of O6-alkylguanine-DNA alkyltransferase activity with protracted temozolomide schedules.

Temozolomide, an oral DNA methylator that inactivates the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGAT), has demonstrated anticancer activity on protracted schedules. Protracted schedules may lead to an 'autoenhancement' of temozolomide's inherent cytotoxic potential by cumulative reduction of the cell's capacity for AGAT-mediated DNA repair and resistance. This study was undertaken to characterise AGAT inactivation and regeneration in the peripheral blood mononuclear cells (PBMCs) of patients treated on two protracted temozolomide schedules. O(6)-alkyl guanine-DNA alkyltransferase activity was measured in the PBMCs of patients treated on two phase I protracted temozolomide studies. Patients were treated daily for either 7 days every 2 weeks (Schedule A) or 21 days every 4 weeks (Schedule B). The effects of various temozolomide doses (75-175 mg m(-2)), treatment duration (7-21 days), and temozolomide plasma levels on AGAT inactivation and regeneration, as well as the relation between AGAT inactivation and toxicity, were studied. O(6)-alkyl guanine-DNA alkyltransferase activity in PBMCs was measured serially in 52 patients. Marked inactivation of AGAT occurred following 7 days of temozolomide treatment, with mean AGAT activity decreasing by 72% (P<0.0001). Similarly, mean AGAT activity decreased by 63 and 73% after 14 and 21 days of treatment, respectively (P<0.001 for both comparisons). O(6)-alkyl guanine-DNA alkyltransferase inactivation was greater after 7 days of treatment with higher doses of temozolomide than lower doses and remained markedly reduced 7 days post-treatment. However, AGAT inactivation following temozolomide treatment for 14 and 21 days was similar at all doses. On the continuous 21-day schedule, AGAT inactivation was significantly greater in patients who experienced severe thrombocytopenia than those who did not (90.3+/-5.5 vs 72.5+/-16.1%, P<0.045). In conclusion, protracted administration of temozolomide, even at relatively low daily doses, leads to significant and prolonged depletion of AGAT activity, which may enhance the antitumour activity of the agent.

Antineoplastic Agents, Alkylating↗
PubMed2003

Hematopoietic stem cell gene therapy: progress toward therapeutic targets.

The concept of hematopoietic stem cell gene therapy is as exciting as that of stem cell transplantation itself. The past 20 years of research have led to improved techniques for transferring and expressing genes in hematopoietic stem cells and preclinical models now routinely indicate the ease with which new genes can be expressed in repopulating stem cells of multiple species. Both modified murine oncoretroviruses and lentiviruses transmit genes into the genome of hematopoietic stem cells and allow expression in the host following transplantation. Using oncoretroviruses, therapeutic genes for severe combined immunodeficiency, common variable gamma chain immunodeficiency, chronic granulomatous disease, Hurler's and Gaucher's Disease have all been used clinically with only modest success except for the patients with immunodeficiency in whom a partial T-cell chimerism has been dramatic. Since stem cell selection in vivo appears important to the therapeutic success of gene transfer, drug resistance selection, most recently using the MGMT gene, has been developed and appears to be safe. Future trials combining a drug resistance and therapeutic gene are planned, as are trials using safety-modified lentiviruses. The therapeutic potential of hematopoietic stem cell gene therapy, particularly given recent advances in stem cell plasticity, remains an exceptionally exciting area of clinical research.

Genetic Therapy↗
PubMed2002

Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH).

Patients with Hurler syndrome (mucopolysaccharidosis type-IH) and metachromatic leukodystrophy (MLD) develop significant skeletal and neurologic defects that limit their survival. Transplantation of allogeneic hematopoietic stem cells results in partial correction of the clinical manifestations. We postulated that some of these defects may be corrected by infusion of allogeneic, multipotential, bone marrow-derived mesenchymal stem cells (MSC). Patients with Hurler syndrome (n = 5) or MLD (n = 6) who previously underwent successful bone marrow transplantation from an HLA-identical sibling were infused with 2-10 x 10(6)/kg MSCs, isolated and expanded from a bone marrow aspirate of the original donor. There was no infusion-related toxicity. In most recipients culture-purified MSCs at 2 days, 30-60 days and 6-24 months after MSC infusion remained of host type. In two patients the bone marrow-derived MSCs contained 0.4 and 2% donor MSCs by FISH 60 days after MSC infusion. In four patients with MLD there were significant improvements in nerve conduction velocities after MSC infusion. The bone mineral density was either maintained or slightly improved in all patients. There was no clinically apparent change in patients' overall health, mental and physical development after MSC infusion. We conclude that donor allogeneic MSC infusion is safe and may be associated with reversal of disease pathophysiology in some tissues. The role of MSCs in the management of Hurler syndrome and MLD should be further evaluated.

Adolescent↗
PubMed2001

MMTV promoter hypomethylation is linked to spontaneous and MNU associated c-neu expression and mammary carcinogenesis in MMTV c-neu transgenic mice.

The erbB family of receptor tyrosine kinases is frequently implicated in neoplasia. Amplification and overexpression of erbB2/neu has been found in 20 to 40% of human breast cancers. Previous studies using MMTV/c-neu transgenic mice have linked rat neu overexpression to mammary tumor development. In this study, we provide evidence that rat neu overexpression in mammary tumors of MMTV/c-neu transgenic mice is always associated with demethylation of the MMTV promoter, whereas the normal mammary glands of these transgenic mice always contain specific methylated regions of the MMTV promoter. In addition, after exposure to N-methyl-N-nitrosourea (MNU), the latency of mammary tumor development is significantly reduced and again is also associated with MMTV promoter demethylation. Thus, the transition from methylation to hypomethylation of the MMTV promoter induces high-level expression of c-neu and appears to be a prerequisite for transformation from normal to malignant mammary epithelium, either spontaneously or after carcinogen exposure. Expression of transgenic c-neu from the demethylated MMTV promoter appears to be an early event that allows outgrowth of mammary epithelium predisposed to malignant transformation.

Alkylating Agents↗
PubMed2001

Overexpression of human O6-alkylguanine DNA alkyltransferase (AGT) prevents MNU induced lymphomas in heterozygous p53 deficient mice.

O6-alkylguanine DNA alkyltransferase (AGT) is a key mechanism in the prevention against MNU induced malignant transformation by removal of O6 methyl guanine (O6mG) adducts. We asked whether heterozygous p53 deficient mice (p53+/-) would be more susceptible to MNU induced lymphomas than wild type mice, and whether O6mG adducts were responsible for this susceptibility. To determine whether MGMT overexpression would be protective, p53+/- mice were bred to human MGMT transgenic mice (MGMT+) and treated with 50 mg/kg MNU. MNU increased the incidence of thymic lymphomas in non-transgenic p53+/- mice from 23% (n=13) to 68% (n=22) and decreased the mean latency from 433 to 106 days (P=0.01 compared to untreated mice). Wild type mice had an incidence of 30% (n=38) and a mean latency of 135 days after MNU. Overexpression of MGMT in the thymus of p53+/- mice significantly reduced the lymphoma incidence from 68 to 28% (n=17) and increased the latency from 106 to 167 days (P=0.003). Similarly, the lymphoma incidence in MGMT+/wild type mice decreased from 30 to 8% (n=12) and the latency increased to 297 days (P=0.2). Loss of the wild type allele was found in only 2/17 lymphomas occurring in p53+/- mice and there were no significant point mutations in exons 5-8 of p53. Furthermore, there was no loss of p53 function in these mice. These data demonstrate that unrepaired O6mG lesions act cooperatively with the reduced p53 dose and lead to lymphomagenesis in p53+/- mice, but AGT overexpression and rapid removal of O6mG adducts is protective.

Alkylating Agents↗
PubMed2001

Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors.

BACKGROUND: Umbilical-cord blood from unrelated donors who are not HLA-identical with the recipients can restore hematopoiesis after myeloablative therapy in children. We studied the use of transplantation of umbilical-cord blood to restore hematopoiesis in adults. METHODS: Sixty-eight adults with life-threatening hematologic disorders received intensive chemotherapy or total-body irradiation and then transplants of HLA-mismatched umbilical-cord blood. We evaluated the outcomes in terms of hematologic reconstitution, the occurrence of acute and chronic graft-versus-host disease (GVHD), relapses, and event-free survival. RESULTS: Of the 68 patients, 48 (71 percent) received grafts of umbilical-cord blood that were mismatched for two or more HLA antigens. Of the 60 patients who survived 28 days or more after transplantation, 55 had neutrophil engraftment at a median of 27 days (range, 13 to 59). The estimated probability of neutrophil recovery in the 68 patients was 0.90 (95 percent confidence interval, 0.85 to 1.0). The presence of a relatively high number of nucleated cells in the umbilical-cord blood before it was frozen was associated with faster recovery of neutrophils. Severe acute GVHD (of grade III or IV) occurred in 11 of 55 patients who could be evaluated within the first 100 days after transplantation. Chronic GVHD developed in 12 of 33 patients who survived for more than 100 days after transplantation. The median follow-up for survivors was 22 months (range, 11 to 51). Of the 68 patients, 19 were alive and 18 of these (26 percent) were disease-free 40 months after transplantation. The presence of a high number of CD34+ cells in the graft was associated with improved event-free survival (P=0.05). CONCLUSIONS: Umbilical-cord blood from unrelated donors can restore hematopoiesis in adults who receive myeloablative therapy and is associated with acceptable rates of severe acute and chronic GVHD.

Acute Disease↗
PubMed2001

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells.

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.

Apoptosis↗
PubMed2001

Phase I clinical and pharmacokinetic study of rebeccamycin analog NSC 655649 given daily for five consecutive days.

PURPOSE: Rebeccamycin analog (NSC 655649) is active against a variety of both solid and nonsolid tumor cell lines. We performed a phase I trial to determine the maximum-tolerated dose (MTD) of rebeccamycin analog when given on a daily x 5 schedule repeated every 3 weeks, characterize the toxicity profile using this schedule, observe patients for antitumor response, and determine the pharmacokinetics of the agent and pharmacodynamic interactions. PATIENTS AND METHODS: Thirty assessable patients received a total of 153 cycles according to the following dose escalation schema: 60, 80, 106, 141, and 188 mg/m(2)/d x 5 days. RESULTS: Grade 2 phlebitis occurred in all patients before the use of central venous access, placed at dose level 4 and higher. Dose-limiting toxicity (DLT), grade 4 neutropenia, occurred at 188 mg/m(2)/d x 5 days in both previously treated and chemotherapy-naive patients. Pharmacokinetic analysis revealed a three-compartmental model of drug elimination and a long terminal half-life (154 +/- 55 hours). The percentage drop in absolute neutrophil count correlates with the area under the curve infinity. The presence of a second peak during the elimination phase as well as a high concentration of NSC 655649 in biliary fluid compared with the corresponding plasma measurement (one patient) is suggestive of enterohepatic circulation. Two partial responses, two minor responses, and six prolonged (> 6 months) cases of stable disease were observed. Of these, three patients with gallbladder cancer and one patient with cholangiocarcinoma experienced either a minor response or a significant period of freedom from progression. CONCLUSION: The recommended phase II dose for NSC 665649 on a daily x 5 every 3 weeks schedule is 141 and 165 mg/m(2)/d for patients with prior and no prior therapy, respectively, with DLT being neutropenia. During this phase I trial, encouraging antitumor activity was been observed.

Adult↗
PubMed2001

Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells.

Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.

Amino Acid Sequence↗
PubMed2001

Systemic deficits in transporter for antigen presentation (TAP)-1 or proteasome subunit LMP2 have little or no effect on tumor incidence.

Some tumor cells have deficits in class I MHC antigen processing, suggesting that T cells exert selective pressure on tumor cells. Previous studies have not revealed increased tumor incidence in mice with deficits in T-cell immunity, including mice lacking TAP1 (a subunit of the transporter for antigen presentation) or LMP2 (a regulated subunit of the 20S proteasome). The incidence of spontaneous tumors in these mice, however, is too low to assess differences in host resistance to tumors. To increase tumor incidence and better assess the role of systemic expression of TAP1 and LMP2 in responses to tumors, TAP1-/- and LMP2-/- mice were bred with p53-/- mice to create TAP1-/-p53-/- and LMP2-/-p53-/- double knockout mice. Lymphomas and sarcomas (malignant fibrous histiocytoma and angiosarcoma) occurred with high incidence in all p53-deficient populations. Tumor incidence and death rate were similar in TAP1-/-p53-/- mice and closely matched control TAP1+/+p53-/- mice. Tumor incidence and death rate were slightly accelerated in LMP2-/-p53-/- mice relative to control LMP2+/+p53-/- mice, but the biological significance of this difference was unclear. The relative incidence of lymphomas vs. sarcomas was not significantly altered by variation in TAP1 or LMP2. In conclusion, systemic absence of TAP1 did not alter tumor incidence, while absence of LMP2 was associated with only a slight acceleration of tumor incidence of uncertain significance. These observations are consistent with other evidence that normal T-cell responses do not effectively limit tumorigenesis. Even though T cells can attack some tumor cells, the ability of tumors to alter their immunogenicity and evade T-cell surveillance may render the native immune system ineffective at providing a rate-limiting barrier to tumorigenesis and preventing cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

MGMT expression in murine bone marrow is a major determinant of animal survival after alkylating agent exposure.

Myelosuppression is commonly observed after alkylating agent chemotherapy due to low levels of O(6)-alkylguanine DNA alkyltransferase protein (AGT) in hematopoietic progenitors. Mice that lack AGT in all organs, O(6)-methylguanine-DNA methyltransferase gene knockout (MGMT(-/-)) mice are extremely hypersensitive to the methylating agent N-methyl-N-nitrosourea (MNU) and exhibit a 10-fold reduction in the LD(90). To determine whether bone marrow damage was the cause of the increased lethality, we transplanted 1 x 10(6) wild-type marrow into MGMT(-/-) mice and MGMT(-/-) marrow into wild-type mice and observed survival after MNU. Lethally irradiated MGMT(-/-) mice given > or = 25 mg/kg MNU 3 weeks after transplant of wild-type cells survived > 30 days (n = 11), whereas this dose was lethal to control MGMT(-/-) mice 9-12 days post treatment (n = 5). Conversely, lethally irradiated wild-type mice transplanted with MGMT(-/-) cells died after only 20-60 mg/kg MNU within 8-12 days (n = 6). No significant toxicities were found in other organs. Additionally, in an in vivo post transplant competition model, wild-type long-term repopulating cells had a > 200-fold competitive survival advantage over MGMT(-/-) cells, and after MNU treatment completely repopulated the mouse when transplanted at only one-tenth the cell number. We also observed a strong selection for transplanted marrow-derived wild-type stromal elements in the MGMT(-/-) background after drug treatment. These data indicate that alkylating agent hypersensitivity of MGMT(-/-) mice results from hematopoietic damage at the stem level. Thus, DNA repair involving AGT in hematopoietic cells is required for normal host survival following exposure to methylating and chloroethylating agents.

Alkylating Agents↗
PubMed2001

G156A MGMT-transduced human mesenchymal stem cells can be selectively enriched by O6-benzylguanine and BCNU.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investigated for a potential therapeutic role as hematopoietic support cells following chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited evidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich marked hMSCs using chemotherapy. We have determined that hMSCs are markedly sensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pretreated with O(6)-benzylguanine (BG) resulting in a more than four-fold decrease in BCNU IC(90). The MFG retroviral vector encoding a bicistronic transcript for green fluorescent protein (GFP) and mutant (G156A)-methylguanine methyltransferase (G156A-MGMT), which encodes O(6)-alkylguanine-DNA alkyltransferase (AGT), conferring, BG plus BCNU resistance, transduced a high percentage of hMSCs. Transduced hMSCs had high expression of GFP and AGT and became significantly resistant to BG and BCNU. Furthermore, the proportion of GFP expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% following BG and BCNU treatment in vitro. Intravenously infused hMSCs were detected in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultured from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse could be recultured at a low frequency, but enriched by BG and BCNU treatment from 0.05 +/- 0.03% to 0.55 +/- 0.4 (p = 0.028, Welch t-test). Our results indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGMT is a powerful selectable gene for a second marker gene in hMSCs. Drug resistance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.

Animals↗
PubMed2001

Temozolomide: the effect of once- and twice-a-day dosing on tumor tissue levels of the DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase.

Temozolomide (TMZ) is a methylating agent of the imidotetrazine class, whose cytotoxic product is O(6)-methylguanine DNA adducts, which initiate a futile recycling of the mismatch repair pathway causing DNA strand breaks and apoptotic cell death in mismatch repair proficient cells. The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT) repairs these adducts in a suicide manner and reduces the cytotoxic action of TMZ. An antitumor threshold is reached when sufficient adducts are formed by TMZ to inactivate AGT. In this study, we evaluated the relation between TMZ dosing and AGT depletion in patients with deep visceral tumors and in peripheral blood mononuclear cells (PBMCs) to determine whether the dose of TMZ was sufficient to inactivate AGT and lead to therapeutic efficacy. To do so, we compared single dose therapy with a novel twice daily regimen in a laboratory correlate-driven Phase I dose escalation study. p.o. bolus dose TMZ 200 mg/m(2) daily times five was compared with the same bolus on day 1 followed by nine doses at 12-h intervals of 50, 75, 90, or 100 mg/m(2). Dose-limiting toxicity in the bid regimen (grade IV thrombocytopenia and neutropenia) was seen at 100 mg/m(2), cumulative dose 1100 mg/m(2), and the maximum tolerated dose was 1010 mg/m(2). The degree of tumor tissue AGT activity depletion measured in biopsies before and on day 5 of therapy varied widely, between 0 (in 3 patients) and 99% (in 1), with the majority of patients (10 of 15) having 52-84% tumor AGT depletion. In contrast, AGT activity in PBMCs fell rapidly during TMZ administration to undetectable levels in all dosage groups on day 5 but did not correlate with tumor AGT depletion. TMZ pharmacokinetics were dose proportional; no accumulation occurred >5-day period in the bid regimen. Two partial responses were seen, lasting 3 and 4 months. Five additional patients achieved prolonged stabilization of disease for 4-6 monthly cycles. This is the first study to document that at maximum tolerated doses, TMZ depletes PBMC AGT but only partially and variably depletes visceral tumor AGT in most patients, even during twice daily dosing. Drug combinations or schedules designed to maximally deplete tumor AGT might improve TMZ efficacy.

Adult↗
PubMed2001

Differential degradation rates of inactivated alkyltransferase in blood mononuclear cells and tumors of patients after treatment with O(6)-benzylguanine.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-alkylating DNA adducts generated by alkylating therapeutic agents. Therefore, AGT activity may be an important marker of tumor and normal tissue sensitivity to chemotherapeutic agents and a predictor for the success of chemotherapeutic regimens. It is rapidly inactivated by O(6)-benzylguanine (BG) that mimics its substrates, O(6)-methylguanine and O(6)-chloroethylguanine DNA adducts. In a Phase I clinical trial, BG was given in increasing doses (from 10 to 120 mg/m(2)) by 1-h infusion. We previously reported depletion of AGT activity, and in this report, we demonstrate the relationship between degradation of BG-inactivated AGT protein and the depletion of AGT activity in peripheral blood mononuclear cells (PBMCs) and tumor samples obtained by computed tomography-guided cutting needle biopsy from patients prior to BG and either 2 or 18 h after BG. In PBMCs, BG inactivated AGT activity by over 95-100% at the end of a 1-h infusion, and depletion was maintained for 18 h. In contrast, AGT protein remained almost unchanged for up to 18 h after BG, suggesting that inactivated AGT proteins remain immunoreactive and are not rapidly degraded in PBMCs. In patient tumor biopsies, AGT activity was depleted approximately 90% 2 h after BG. Tumor AGT protein levels were reduced to approximately 40% of pretreatment values when detected by either Western blot or immunohistochemistry staining. In tumor samples obtained 18 h after BG, >95% inactivation of tumor AGT activity was observed at BG doses of 36-80 mg/m(2), and complete depletion of tumor AGT activity occurred at 120 mg/m(2) BG. However, residual AGT protein (5-10% of baseline) was detectable in all tumor samples. Therefore, the degradation of BG-inactivated AGT protein appeared to be much more rapid in tumors than that in PBMCs, which may impact on AGT regeneration rates as well. Because degradation of BG-inactivated AGT takes place slowly, antibody-based measurements of AGT protein correlate poorly with depletion of AGT activity immediately after BG. Thus, biochemical activity measurements remain the appropriate monitor of AGT during therapeutic modulation. These data provide the first and conclusive evidence of differential degradation rates of inactivated AGT in PBMCs and tumors of patients after treatment with BG and suggest that immunoreactive AGT measurements in PBMCs are a poor surrogate for AGT activity in tumor tissue.

Biopsy↗
PubMed2001

Sequential tumor biopsies in early phase clinical trials of anticancer agents for pharmacodynamic evaluation.

PURPOSE: In the setting of target-based anticancer drug development, it is critical to establish that the observed preclinical activity can be attributed to modulation of the intended target in early phase trials in human subjects. This paradigm of target modulation allows us to determine a Phase II or III dose (optimal biochemical/biological modulatory dose) that may not necessarily be the maximum tolerated dose. A major obstacle to target-based (often cytostatic) drug development has been obtaining relevant tumor tissue during clinical trials of these novel agents for laboratory analysis of the putative marker of drug effect. EXPERIMENTAL DESIGN: From 1989 to present, we have completed seven clinical trials in which the end point was a biochemical or biological modulatory dose in human tumor tissues (not surrogate tissue). Eligibility enrollment required that patients have a biopsiable lesion either with computerized tomography (CT) guidance or direct visualization and consent to sequential (pre and posttreatment) biopsies. RESULTS: A total of 192 biopsies were performed in 107 patients. All but 8 patients had sequential pre and posttreatment biopsies. Seventy-eight (73%) of the 107 patients had liver lesion biopsies. In eight patients, either one or both biopsies contained insufficient viable tumor tissue or no tumor tissue at all for analysis. Of a total of 99 patients in whom we attempted to obtain paired biopsies, a total of 87 (88%) were successful. Reasons for failure included patient refusal for a second biopsy (n = 2), vasovagal reaction with first biopsy precluding a second biopsy (n = 1), subcapsular hepatic bleeding (n = 1), and most commonly obtaining necrotic tumor, fibrous, or normal tissue in one of the two sequential biopsies (n = 8). CONCLUSIONS: This is the first and largest reported series demonstrating that with adequate precautions and experience, sequential tumor biopsies are feasible and safe during early phase clinical trials.

Animals↗
PubMed2000

Limiting numbers of G156A O(6)-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection.

The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (triangle upMGMT) transduced LTRC present in 5 x 10(4) to 100 x 10(4) marrow cells infused into nonmyeloablated mice by the administration of O(6)-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, triangle upMGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, triangle upMGMT(+) CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% triangle upMGMT(+) CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows triangle upMGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that triangle upMGMT may be a potent, dominant, selectable marker for use in dual gene therapy.

Animals↗
PubMed2000

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen.

PURPOSE AND METHODS: The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. RESULTS: We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O6-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. CONCLUSION: Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance.

Adaptor Proteins, Signal Transducing↗