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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.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.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).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.

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.

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 occurred5-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.

Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent O6-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T)) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2. In tumor tissue, AGT depletion86% of baseline was demonstrated at all doses tested. Residual tumor AGT activity, present 18 h after BG doses of 10-80 mg/m2, was eliminated at the 120 mg/m2 dose and is thus the BMD(T) of BG. BG pharmacokinetics are characterized by the rapid, dose-independent clearance of BG from plasma Metabolism of BG to its biologically active metabolite, 8-oxo-benzylguanine (8-oxo-BG), was found. The t(1/2) of 8-oxo-BG is longer than BG. Plasma concentrations of 8-oxo-BG well above 200 ng/ml 18 h after the end of the BG infusion were observed at the highest dose levels tested and appeared to correlate with depletion of AGT activity to undetectable levels in tumor tissue. AGT activity in peripheral blood mononuclear cells at baseline did not correlate with tumor tissue AGT activity. Depletion of AGT activity to undetectable levels in peripheral blood mononuclear cells occurred at lower doses and was not a reliable predictor for tumor tissue depletion. No serious side effects were observed with administration of BG alone or in combination with 13 mg/m2 1,3-bis(2-chloroethyl)-1-nitrosourea. This is the first clinical study in which biochemical analyses from pre- and posttreatment tumor biopsies have been used as an efficacy end point for the clinical development of an anticancer agent. From our tumor tissue biopsy data, we have established that a BG dose of 120 mg/m2 infused over 1 h should be used in Phase II clinical trials.

A 37-year-old woman with Philadelphia chromosome-positive acute lymphoblastic leukemia received allogeneic bone marrow transplants after first and second relapses from different HLA-identical brothers. After a third relapse, blood progenitor cells from one of the brothers were infused after six doses of cytarabine 1.5 g/m2 in an attempt to induce a graft-versus-leukemia effect. Seven weeks after the infusion, the patient developed delayed but severe acute graft-versus-host disease (GVHD) including the unusual manifestation of conjunctival involvement.

A patient with Paget's disease of bone and primary hyperparathyroidism is described. It has been suggested that a metabolite from pagetic tissue might stimulate parathyroid cell function. To test this, serum parathyroid hormone (iPTH) levels were measured by radio immunometric assay in 106 patients with Paget's disease of bone and 139 control subjects. There was no significant difference between the mean levels of the two groups. Of five sera with elevated PTH levels QUOSO extraction revealed an interfering substance in 3/5 from control patients tested and extractable iPTH in 2/2 from Paget's patients. One of the latter had renal failure. The results suggest that increased parathyroid function is unlikely to be a primary event in the genesis of Paget's disease of bone.

A patient is described with a malignant carcinoid tumour of the ileum with nodal secondaries causing mesenteric vessel occlusion and ileal infarction.

Walker 256 carcinosarcoma cells have been shown to respond in vitro to two chemotactic factors: one derived from the trypsinized fifth component of complement, and the other derived from resorbing bone. The effects of inhibited microtubule and microfilament function on the ability of tumor cells to uindergo both random and directed migration in response to these factors were examined. Directed tumor cell migration was inhibited by colchicine and vinblastine, agents that interfere with microtubule assembly., Lumicolchicine, a structural analog of colchicine that does not bind to microtubules, did not inhibit tumor cell migration. Cytochalasin B, which interferes with microfilament function, also inhibited tumor cell migration in vitro. Colchicine inhibited random or spontaneous cell movement as well as directed migration. These experiments indicated that random and directed tumor cell migrations were dependent on microtubules and microfilaments, and interference with the function of these subcellular organelles inhibited tumor cell movement.