Carmen J. Allegra

DNA Mismatch Repair Status and Colon Cancer Recurrence and Survival in Clinical Trials of 5-Fluorouracil-Based Adjuvant Therapy

BACKGROUND Approximately 15% of colorectal cancers develop because of defective function of the DNA mismatch repair (MMR) system. We determined the association of MMR status with colon cancer recurrence and examined the impact of 5-fluorouracil (FU)-based adjuvant therapy on recurrence variables. METHODS We included stage II and III colon carcinoma patients (n = 2141) who were treated in randomized trials of 5-FU-based adjuvant therapy. Tumors were analyzed for microsatellite instability by polymerase chain reaction and/or for MMR protein expression by immunohistochemistry to determine deficient MMR (dMMR) or proficient MMR (pMMR) status. Associations of MMR status and/or 5-FU-based treatment with clinicopathologic and recurrence covariates were determined using χ(2) or Fisher Exact or Wilcoxon rank-sum tests. Time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) were analyzed using univariate and multivariable Cox models, with the latter adjusted for covariates. Tumors showing dMMR were categorized by presumed germline vs sporadic origin and were assessed for their prognostic and predictive impact. All statistical tests were two-sided. RESULTS In this study population, dMMR was detected in 344 of 2141 (16.1%) tumors. Compared with pMMR tumors, dMMR was associated with reduced 5-year recurrence rates (33% vs 22%; P < .001), delayed TTR (P < .001), and fewer distant recurrences (22% vs 12%; P < .001). In multivariable models, dMMR was independently associated with delayed TTR (hazard ratio = 0.72, 95% confidence interval = 0.56 to 0.91, P = .005) and improved DFS (P = .035) and OS (P = .031). In stage III cancers, 5-FU-based treatment vs surgery alone or no 5-FU was associated with reduced distant recurrence for dMMR tumors (11% vs 29%; P = .011) and reduced recurrence to all sites for pMMR tumors (P < .001). The dMMR tumors with suspected germline mutations were associated with improved DFS after 5-FU-based treatment compared with sporadic tumors where no benefit was observed (P = .006). CONCLUSIONS Patients with dMMR colon cancers have reduced rates of tumor recurrence, delayed TTR, and improved survival rates, compared with pMMR colon cancers. Distant recurrences were reduced by 5-FU-based adjuvant treatment in dMMR stage III tumors, and a subset analysis suggested that any treatment benefit was restricted to suspected germline vs sporadic tumors.

Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer.

The efficacy and toxicity of leucovorin 500 mg/m2 administered intravenously (IV) over 30 minutes daily for five days followed in one hour by fluorouracil (5-FU) 375 mg/m2 administered IV daily for five days, each given every 3 weeks, was assessed in 54 previously treated patients with metastatic breast cancer. An overall objective response rate of 24% was achieved (95% confidence interval, 13% to 38%), with an additional 56% of patients maintaining stable disease. Eleven of 12 patients who responded had received previous 5-FU therapy. Toxicity of this regimen included grade 3 diarrhea in 13%, grade 3 or 4 mucositis in 33%, grade 3 or 4 granulocytopenia in 65%, and grade 3 or 4 thrombocytopenia in 19%. Delay of treatment was required for hematologic toxicity in 44 patients. Thirty-eight patients required dose reductions due to toxicity. Biochemical evaluation of tumor biopsy specimens obtained from 17 patients used as their own controls with and without leucovorin was performed. These studies reveal an increased stabilization of the 5-fluorodeoxyuridylate (FdUMP)-thymidylate synthase (TS) folate ternary complex with the addition of leucovorin. There was a 71% +/- 14% occupancy or inhibition of the enzyme with the use of both 5-FU and leucovorin, v 30% +/- 13% for 5-FU alone (P2 less than .037). The percent TS bound in responding patients was substantially higher than in those patients with progressive disease. Finally, the mean total tumor TS pre-therapy in seven patients was 31 fmol/mg compared with a mean of 81 fmol/mg in these same seven patients 24 hours after therapy. This 2.6-fold increase suggests that there is an induction of the enzyme, TS, with 5-FU treatment.

Regulation of Thymidylate Synthase in Human Colon Cancer Cells Treated with 5-Fluorouracil and Interferon-Gamma

The effects of fluorouracil (5-FU) and interferon-gamma (IFN-gamma) on the regulation of thymidylate synthase (TS) gene expression were investigated in the human colon cancer H630 cell line. By Western immunoblot analysis, TS protein levels in H630 cells were increased 3-, 5.5-, 5-, and 2.5-fold after 8-, 16-, 24-, and 36-hr exposure to 1 microM 5-FU, respectively. When H630 cells were exposed to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, increases in TS protein up to 5.5-fold were observed. A 24-hr exposure to 1 microM 5-FU resulted in a 4.5-fold increase in the level of TS protein, whereas in 5-FU/IFN-gamma-treated cells TS protein was increased by only 1.8-fold, compared with control cells. IFN-gamma treatment alone did not affect TS protein levels, relative to control. Northern blot analysis revealed no changes in TS mRNA levels when H630 cells were exposed either to 1 microM 5-FU for 8-36 hr, to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, or to the combination of 5-FU and IFN-gamma. Pulse-labeling studies with [35S]methionine demonstrated a 3.5-fold increase in net synthesis of TS in cells treated with 1 microM 5-FU, whereas the level of newly synthesized TS increased only 1.5-fold in cells treated with 5-FU/IFN-gamma, compared with control cells. Pulse-chase studies revealed that the half-lives of TS protein in control and 5-FU-treated cells were equivalent. These findings demonstrate that the increase in TS protein after 5-FU exposure and the subsequent inhibitory effect of IFN-gamma on TS protein expression are both regulated at the post-transcriptional level.

A phase III trial comparing mFOLFOX6 to mFOLFOX6 plus bevacizumab in stage II or III carcinoma of the colon: Results of NSABP Protocol C-08

LBA4 Background: The primary aim of this two-arm randomized prospective study was to determine whether mFOLFOX6 plus bevacizumab (mFF6+B) would prolong disease-free survival (DFS) compared to mFOLFOX6 (mFF6) alone. METHODS Between September 2004 and October 2006, 2,672 patients with follow-up (1,338 and 1,334 in respective arms) with stage II (24.9%) or III carcinoma of the colon were randomized to receive either mFF6 (oxaliplatin 85 mg/m2 IV d1, leucovorin 400 mg/m2 IV d1, 5-FU 400 mg/m2 IV bolus d1, and 5-FU 2400 mg/ m2 CI over 46 hrs (d1+2) q14d × 12 cycles) or mFF6+B (same mFF6 regimen + bevacizumab 5 mg/kg IV q 2 wks × 1 yr). The primary end point was DFS. Events were defined as first recurrence, second primary cancer, or death. RESULTS The median follow-up for patients still alive was 36 months. The hazard ratio (HR: FF6+B vs. mFF6) was 0.89; 95% CI (0.76=1.04); p=0.15. Data censored at intervals disclosed an initial benefit for bevacizumab that diminished over time: The smoothed estimate of the DFS HR over time indicated that bevacizumab significantly reduced the risk of a DFS event during the interval from 0.5 to 1.0 year. There was no evidence that patients receiving bevacizumab had a worse DFS compared to those receiving mFF6 alone following treatment. CONCLUSIONS The addition of bevacizumab to mFF6 did not result in an overall statistically significant prolongation in DFS. There was a transient benefit in DFS during the one-year interval that bevacizumab was utilized. Consideration may be given to clinical trials assessing longer duration of bevacizumab administration. Supported by PHS grants U10CA-12027, -69974, -37377, and -69651 from the NCI and a grant from Genentech, Inc. [Table: see text] No significant financial relationships to disclose.

Prognostic importance of thymidylate synthase expression in early breast cancer.

PURPOSE To assess the prognostic importance of thymidylate synthase (TS) expression in breast tumors of patients with early-stage breast cancer, and to determine whether the benefit of chemotherapy (CT) is associated with TS expression. PATIENTS AND METHODS The level of TS expression was evaluated in 210 node-negative and 278 node-positive patients enrolled onto Trial V of the International Breast Cancer Study Group ([IBCSG] formerly the Ludwig Breast Cancer Study Group) with a median follow-up time of 8.5 years. TS expression was assessed using the immunohistochemical method with the monoclonal antibody TS 106 on paraffin-embedded tissue specimens. RESULTS High TS expression was associated with a significantly worse prognosis in node-positive but not in node-negative breast cancer patients. Twenty-seven percent of node-positive patients with high TS expression were disease-free at 10 years, compared with 44% of node-positive patients with low TS expression (P = .03). Forty-one percent of patients with node-positive high-TS-expressing tumors were alive after 10 years, compared with 49% of those with low TS expression (P = .06). The association between TS and disease-free survival (DFS) and overall survival (OS) was independent of other prognostic factors such as tumor size, tumor grade, nodal status, vessel invasion, estrogen receptor (ER)/ progestin receptor (PR) status, c-erb B-2, or Ki-67 expression. In node-positive patients, six cycles of standard adjuvant cyclophosphamide, methotrexate, and fluorouracil ([5-FU] CMF) CT improved DFS and OS compared with one cycle of perioperative CMF therapy. The magnitude of this benefit was greatest in patients whose tumors had high TS expression (P < .01 for DFS; P < .01 for OS). Node-negative patients demonstrated no difference in outcome to CT based on TS expression; however, the power to detect differences was limited by the small number of events in this group. CONCLUSION In early-stage breast cancer, high TS expression is associated with a significantly worse prognosis in node-positive patients. Node-positive patients with high TS levels demonstrate the most significant improvement in DFS and OS when treated with six cycles of conventional adjuvant CMF therapy.

A phase I vaccine trial with peptides reflecting ras oncogene mutations of solid tumors.

Mutations in the ras genes occur in 20% of all human cancers. These genes, in turn, produce mutated proteins that are unique to cancer cells, rendering them distinguishable from normal cells by the immune system. Thus, mutated Ras proteins may form potential targets for immune therapy. We conducted a phase I/pilot clinical trial in patients with advanced cancers to test the toxicity and the ability to induce an immune response by vaccination with 13-mer mutated Ras peptides reflecting codon 12 mutations. These peptides corresponded to each of the patients own tumor Ras mutation. Patients were vaccinated monthly x3 subcutaneously with the specific Ras peptide along with Detox adjuvant (RiBi ImmunoChem Research, Inc., Hamilton, MT, U.S.A.) at one of five different peptide dose levels (100, 500, 1,000, 1,500, and 5,000 micrograms). Three out of 10 evaluable patients generated a mutant Ras specific CD4+ and/or CD8+ T-cell immune response. The CD8+ cytotoxic cells specific for Gly to Val mutation at codon 12 were capable of lysing an HLA-A2-matched tumor cell line carrying the corresponding mutant but not the wild-type ras gene. The treatment has been well tolerated with no evidence of serious acute or delayed systemic side effects on any of the five dose levels. We demonstrated that we can generate in cancer patients specific T-lymphocyte responses that detect single amino acid differences in Ras oncoproteins. Neither the immune responses nor the minor side effects seen were found to be dose dependent. This approach may provide a unique opportunity for generating a tumor-directed therapy. Also, in vitro stimulation of these cells with the corresponding peptide generated specific T-cell lines that could be used for adoptive immune therapy.

Tumor marker expression is predictive of survival in patients with esophageal cancer

BACKGROUND This study was designed to determine the prognostic value of immunohistochemical tumor marker expression in a population of patients with node-negative esophageal cancer treated with complete resection alone. METHODS Resection specimens were collected from 61 patients with node-negative T1 (n = 31), T2 (n = 14), and T3 (n = 16) esophageal cancer. A panel of 10 tumor markers was chosen for immunohistochemical analysis, based on associations with differing oncologic mechanisms: apoptosis (p53), growth regulation (transforming growth factor-alpha, epidermal growth factor receptor, and Her2-neu), angiogenesis (factor VIII), metastatic potential (CD44), platinum resistance (p-glycoprotein and metallothionein), 5-fluorouracil resistance (thymidylate synthetase), and carcinogenic detoxification (glutathione S-transferase-pi). RESULTS Complete resection was performed in all patients (44 adenocarcinoma, 17 squamous cell carcinoma), with no operative deaths. Multivariable analysis demonstrated a significant relationship between cancer-specific death and the following variables: low-level P-gp expression (p = 0.004), high-level expression of p53 (p = 0.04), and low-level expression of transforming growth factor-alpha (p = 0.03). In addition, the number of involved tumor markers present was strongly predictive of negative outcome (p = 0.0001). CONCLUSIONS This study supports the prognostic value of immunohistochemical tumor markers, specifically the expression pattern of P-gp, p53, and transforming growth factor-alpha, in patients with esophageal carcinoma treated with complete resection alone.

Phase I and pharmacologic study of 9-aminocamptothecin given by 72-hour infusion in adult cancer patients.

PURPOSE To conduct a phase I and pharmacologic study of the new topoisomerase I inhibitor, 9-aminocamptothecin (9-AC). PATIENTS AND MATERIALS A 72-hour infusion of 9-AC was administered every 14 days to 48 solid-tumor patients at doses of 5 to 59 microg/m2/h without granulocyte colony-stimulating factor (G-CSF) and 47 to 74 microg/m2/h with G-CSF. RESULTS Without G-CSF, two of eight patients who received 47 microg/m2/h had dose-limiting neutropenia in their initial cycle, as did both patients who received 59 microg/m2/h (with a platelet count < 25,000/microL in one). With G-CSF, zero of seven patients treated with 47 microg/m2/h had dose-limiting neutropenia in their first cycle, while dose-limiting neutropenia occurred in six of 14 patients (with platelet count < 25,000/microL in five) entered at 59 microg/m2/h. Among 39 patients entered at > or = 25 microg/m2/h 9-AC with or without G-CSF, fatigue, diarrhea, and nausea/vomiting of grade 2 severity ultimately occurred in 54%, 30%, and 38%, respectively, while grade 3 toxicities of each type occurred in 8% of patients. Steady-state 9-AC lactone concentration (Css) increased linearly from 0.89 to 10.6 nmol/L, and correlated strongly with leukopenia ( r = .85). CONCLUSION The recommended phase II dose of 9-AC given by 72-hour infusion every 2 weeks is 35 microg/m2/h without G-CSF or 47 microg/m2/h with G-CSF support. Dose escalation in individual patients may be possible according to their tolerance.

Potent antipneumocystis and antitoxoplasma activities of piritrexim, a lipid-soluble antifolate.

Piritrexim, a lipid-soluble antifolate, was evaluated for its activity against Pneumocystis carinii and Toxoplasma gondii. The concentration of piritrexim needed to inhibit 50% of the catalytic activity of P. carinii dihydrofolate reductase (DHFR) was 19.3 nM, and that for T. gondii DHFR was 17.0 nM, concentrations that were 40- to over 1,000-fold less than those needed for the inhibition of activity by trimethoprim and pyrimethamine, the antifolates conventionally used in treating these organisms. Piritrexim was able to inhibit replication of T. gondii in a mouse peritoneal macrophage model at concentrations of 0.1 to 1.0 microM. Leucovorin, a reduced folate that can bypass the inhibition of DHFR by antifols in mammalian cells but not in protozoa, did not affect the ability of piritrexim to inhibit T. gondii replication. The addition of sulfadiazine, which alone was ineffective, to piritrexim allowed inhibition of T. gondii replication at lower concentrations of piritrexim than when piritrexim was used alone. These results suggest that piritrexim, alone or combined with a sulfonamide, may be a highly potent antitoxoplasma and antipneumocystis agent that could provide major pharmacologic and clinical advantages over available agents.

Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate.

Trimetrexate, a highly lipid-soluble quinazoline antifolate now undergoing trials as an anticancer agent, was found to be a potent inhibitor of the dihydrofolate reductase (DHFR) isolated from Toxoplasma gondii. The concentration required for 50% inhibition of protozoal DHFR was 1.4 nM. As an inhibitor of this enzyme, trimetrexate was almost 600-fold (amount of antifolate required to inhibit catalytic reaction by 50%) and 750-fold (inhibition constant) more potent than pyrimethamine, the DHFR inhibitor currently used to treat toxoplasma infection. When the protozoan was incubated with 1 microM trimetrexate, the drug rapidly reached high intracellular concentrations. Since toxoplasma organisms lack a transmembrane transport system for physiologic folates, host toxicity can be prevented by co-administration of the reduced folate, leucovorin, without reversing the antiprotozoal effect. The effectiveness of trimetrexate against toxoplasma was demonstrated both in vitro and vivo. Proliferation of toxoplasma in murine macrophages in vitro was completely inhibited by exposure of these cells to 10(-7) M trimetrexate for 18 h. When used alone, trimetrexate was able to extend the survival of T. gondii-infected mice.