Dose and dose intensity as determinants of outcome in the adjuvant treatment of breast cancer. The Cancer and Leukemia Group B

BACKGROUND: Both total dose and dose intensity of adjuvant chemotherapy are postulated to be important variables in the outcome for patients with operable breast cancer. The Cancer and Leukemia Group B study 8541 examined the effects of adjuvant treatment using conventional-range dose and dose intensity in female patients with stage II (axillary lymph node-positive) breast cancer. METHODS: Within 6 weeks of surgery (radical mastectomy, modified radical mastectomy, or lumpectomy), 1550 patients with unilateral breast cancer were randomly assigned to one of three treatment arms: high-, moderate-, or low-dose intensity. The patients received cyclophosphamide, doxorubicin, and 5-fluorouracil on day 1 of each chemotherapy cycle, with 5-fluorouracil administration repeated on day 8. The high-dose arm had twice the dose intensity and twice the drug dose as the low-dose arm. The moderate-dose arm had two thirds the dose intensity as the high-dose arm but the same total drug dose. Disease-free survival and overall survival were primary end points of the study. RESULTS: At a median follow-up of 9 years, disease-free survival and overall survival for patients on the moderate- and high-dose arms are superior to the corresponding survival measures for patients on the low-dose arm (two-sided P<.0001 and two-sided P = .004, respectively), with no difference in disease-free or overall survival between the moderate- and the high-dose arms. At 5 years, overall survival (average +/- standard error) is 79% +/- 2% for patients on the high-dose arm, 77% +/- 2% for the patients on the moderate-dose arm, and 72% +/- 2% for patients on the low-dose arm; disease-free survival is 66% +/- 2%, 61% +/- 2%, and 56% +/- 2%, respectively. CONCLUSION: Within the conventional dose range for this chemotherapy regimen, a higher dose is associated with better disease-free survival and overall survival.     

A successful system of scientific data audits for clinical trials. A report from the Cancer and Leukemia Group B

OBJECTIVE: To report on data collected during on-site audits of source documents in the Cancer and Leukemia Group B (CALGB). DESIGN: A retrospective review of audit reports in four audit cycles. SETTING: A cooperative group of institutions conducting clinical trials in cancer treatment. PARTICIPANTS: Patients taking part in clinical trials at collaborating CALGB institutions, members of the CALGB Data Audit Committee, and group chairmen of CALGB. MAIN OUTCOME MEASURE: The results of 691 institutional audits conducted by the CALGB in 1982 through 1992 with comparisons of main CALGB institutions vs affiliates. RESULTS: In four full reviews of all participating institutions in the CALGB, 3787 patients have had their on-site medical records compared with data submitted to the CALGB Data Management Center. Compliance with federal regulations for oversight by an institutional review board improved from a deficiency rate of 28.0% among the main institutions and 49.6% of the affiliate institutions in the first audit cycle to respective figures of 13.3% and 28.2% in the fourth cycle. Consent form deficiencies also dropped overall from 18.5% in the first cycle to 3.9% in the fourth. Patient eligibility was verified by auditors in 94.5%, and assessment of tumor changes in response to treatment was verified in 96.4% in the fourth cycle; both figures were only slightly lower in the first cycle. Two instances of scientific impropriety were discovered for a rate of only 0.28% of all audits. Both occurred prior to 1984, and none have occurred since. Major protocol deviations in drug dosing have held steady at about 11% over four audit cycles. Over the 11-year period of audits, three main institutions and 96 affiliate institutions have discontinued CALGB membership due solely, or at least partly, to unfavorable audit results. CONCLUSION: Scientific improprieties have occurred very rarely in clinical trials conducted by the CALGB. Protocol compliance in assessing patient eligibility and tumor responses has been high. Attention to administrative matters of consent forms, institutional review board approval, and ancillary data submission has measurably improved in the CALGB, which is at least partly due to the pressure from this on-site peer review of investigator performance.     

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using ³²P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.     

Constitutive achlorhydria in mucolipidosis type IV

Mucolipidosis type IV is an autosomal recessive lysosomal storage disease of unknown etiology that causes severe neurological and ophthalmological abnormalities. In an attempt to obtain insight into the nature of the metabolic abnormality in this disorder, we prospectively evaluated 15 consecutive patients, aged 2 to 23 years, over a period of 22 months. The finding of iron deficiency in some of the patients led us to the discovery that all patients but one had markedly elevated blood gastrin levels. None had vitamin B12 deficiency. Gastroscopy in three patients showed normal gross appearance of the mucosa in two patients, 4 and 7 years old, and mucosal atrophy in a 22-year-old. Parietal cells were present in normal numbers and contained large cytoplasmic inclusions that were confirmed immunohistochemically to be lysosomal in nature. Other gastric epithelial cells appeared normal. Parietal cells contained very few tubulovesicular membranes, suggesting cellular activation, whereas apical canaliculi appeared relatively nonactivated. Both subunits of the parietal cell H+/K+-ATPase were present, and both partially colocalized with f-actin at the apical membrane. We conclude that patients with mucolipidosis type IV are constitutively achlorhydric and have partially activated parietal cells. We hypothesize that the defective protein in this disease is closely associated with the final stages of parietal cell activation and is critical for a specific type of cellular vacuolar trafficking between the cytoplasm and the apical membrane domain.     

Characterization of lidocaine-specific T cells

To investigate the cellular immune response to the drug lidocaine, we generated T cell lines and clones from the peripheral blood of four patients with proven allergy to lidocaine. The patients had contact dermatitis after topical application of lidocaine, and local swelling or generalized erythema exudativum multiforme after submucosal/subcutaneous injection of lidocaine. Two of three lidocaine-specific T cell lines were oligoclonal and one even became monoclonal, while the simultaneously analyzed immune response to tetanus toxoid was polyclonal. The lidocaine-specific T cell lines cross-reacted to mepivacaine, but not to other local anesthetics (bupivacaine, procaine, oxybuprocaine, and tetracaine). The majority of reactive T cells belonged to the CD4 cell lineage and were MHC class II restricted, but cloning also revealed some MHC class I-restricted CD8+ clones. A total of 2 of 56 lidocaine-specific T cell clones were CD4-CD8- and expressed TCR-gammadelta. The majority of 13 analyzed CD4 clones produced a rather polarized cytokine pattern, with a dominance of Th2-like cytokines showing a high IL-5 production. In addition, three CD4+ and all CD8+ (n = 7) clones secreted high IFN-gamma and low levels of IL-5/IL-4 (Th1-like). The data illustrate that a drug that sensitizes via the skin elicits a heterogeneous T cell response. The high IL-5 production and the participation of specific CD4+CD8+ and even gammadelta+ T cells appear to be distinguishing features of this hapten-specific immune response.     

Lower limb paralysis induced in mice by a temperature-sensitive mutant of Moloney leukemia virus

A temperature-sensitive mutant of Moloney murine leukemia virus defective in an early function and injected into newborn mice produced lower limb paralysis. Susceptible mice were inbred strains CFW/D, CBA/H, C3H/Bi/Ka, and outbred NIH Swiss stock. Inbred W/Fu rats and C57BL/Ka mice did not develop the paralysis, though the latter were infected with virus; the sera from these mice produced paralysis in susceptible CFW mice.     

Gadolinium enhancement: improved MRI detection of retinoblastoma extension into the optic nerve

We performed T1-, T2-, proton density-weighted, and T1-weighted gadolinium-enhanced MRI on 24 patients with retinoblastoma, using a 1.5 T superconducting unit and head and orbital surface coil imaging. All patients underwent a complete ophthalmologic examination, including B-scan ultrasonography. CT was performed on 10 of 24 patients. Pathologic correlation was obtained in 18 patients who required enucleation. Contrast-enhanced T1-weighted MRI with fat suppression was the sequence most sensitive to optic nerve extension and provided the greatest differentiation between tumor and uninvolved extrascleral tissue. Retinoblastoma demonstrated contrast enhancement.