John S. Kovach

A controlled study of combined 1,3‐bis‐(2‐chloroethyl)‐1‐nitrosourea and 5‐fluorouracil therapy for advanced gastric and pancreatic cancer

Treatment with the combination of 1, 3‐bis‐(2‐chloroethyl)‐1‐nitrosourea (BCNU) and 5‐fluorouracil (5‐FU) was compared to therapy with each drug used alone in a prospective randomized study of 167 patients with advanced adenocarcinoma of the stomach and pancreas. At dosages producing comparable degrees of hematologic toxicity, the combination was associated with the highest rate of objective response, 41.3% in carcinoma of the stomach and 33.3% in carcinoma of the pancreas. The corresponding rates of objective response with 5‐FU alone were 28.6% and 16.1%; and with BCNU alone 17.4% and 0%. In pancreatic carcinoma there was no discernible difference in survival among patients in each treatment arm. In gastric carcinoma, however, both 5‐FU and die combination produced an increase in survival when compared to BCNU alone, and the combination of 5‐FU and BCNU produced an increase in long‐term survival compared to 5‐FU alone.

A phase I clinical trial of recombinant human tumor necrosis factor

We performed a Phase I assessment of recombinant human tumor necrosis factor (rTNF‐α) in 27 patients with advanced solid neoplasms. Therapy consisted of a 30‐minute intravenous (IV) infusion on days 1 through 5, every 2 to 3 weeks. Daily doses ranged from 5 μg/m2 to 200 μg/m2. Dose‐limiting sequelae were hypotension, rigors, and phlebitis. Transient fatigue and fever (median, 38°C) were not clearly dose‐related between 5 μg/m2/d and 150 μg/m2/d. Other reversible complications in three patients included transient leukopenia (leukocyte count, 1.3,1.2 × 103/μl in two patients) at a dose of 5 μg/m2/d and 150 μg/m2/d, respectively; and thrombocytopenia (leukocyte count, 73 × 103/μl) at 10 μg/m2/d. Among 22 patients with initial and subsequent differential counts, the median number of eosinophils at the commencement of therapy was 182 cells/μl compared with a subsequent median of 462 cells/μl. We also detected hypertriglyceridemia in all patients. The median baseline increased from 93 mg/dl (range, 56 to 219 mg/dl) to 203 mg/dl (range, 94 to 454 mg/dl). From our experience, a clinically manageable outpatient regimen for Phase II trials consists of rTNF‐α (150 μg/m2) followed by a 1‐hour IV infusion of 500 ml of normal saline to abrogate hypotension daily for 5 days every 2 weeks for four cycles, then every 3 weeks thereafter to facilitate recovery from constitutional sequelae.

Overexpression and mutations of p53 in metastatic malignant melanomas

Alterations of the p53 tumor suppressor gene are the most frequent genetic abnormalities in human malignancies, but the role of p53 in the etiology of malignant melanomas is unclear. Fifty unselected malignant melanomas were analyzed for p53 overexpression by immunohistochemistry using 3 monoclonal antibodies (MAbs). Fifteen tumors (29.4%) showed positive staining with at least 2 different antibodies. In the first 20 consecutive tumors exons 5–9 and adjacent splice sites of the p53 gene were analyzed by genomic sequencing. There were 4 mutations in 20 metastatic melanomas. Three of 4 mutations were C:G → T:A transitions. A search of our database of p53 mutations revealed that out of 8 p53 mutations reported by others, 4 are C:G → T:A transitions at dipyrimidine sites, and one is a tandem CC → TT mutation. This mutational pattern is comparable with the pattern of p53 mutations in squamous cell and basal cell carcinomas of the skin and is related to exposure to ultraviolet B (UV‐B) wavelength radiation. Taken together with a predominance of UV‐induced mutations in the CDKN2/p16 gene demonstrated in melanoma cell lines, our data support a role of sunlight exposure in the etiology of malignant melanoma. The low frequency of p53 mutants in melanomas compared with other types of skin cancers suggests that although mutations in this gene are likely to be involved in the development of some malignant melanomas, they do not play as large a role as in squamous and basal cell carcinomas of the skin.

Novel pattern of p53 gene mutations in an American black cohort with high mortality from breast cancer

The pattern of acquired mutations in the p53 gene can be used to study differences in factors contributing to carcinogenesis. We investigated mutations in exons 5-9 and adjacent intronic regions in 47 breast cancers of black women from Michigan, a population with the highest breast-cancer mortality in the US. The 16 mutations detected differed from those of other populations. In particular, the black women had an excess of A:T-->G:C transitions compared with rural white US midwest women. While the causes of the different pattern of acquired mutation remain to be determined, this molecular epidemiological approach detects the consequences of mutagenic processes in specific populations. Mutation patterns will constrain hypotheses to mechanisms consistent with the observed biochemical alterations.

A prospective trial of midwest breast cancer patients: A p53 gene mutation is the most important predictor of adverse outcome

Several retrospective studies have suggested p53 gene mutation as an adverse prognostic indicator in breast cancer patients, based on a selective growth advantage of p53 mutant cancer cells and their presumed resistance to current adjuvant therapy regimens. A cohort of 90 Caucasian midwestern breast cancer patients was analyzed prospectively (60 months of follow‐up) with a rigorous mutation detection methodology. The presence of a p53 gene mutation was the single most adverse prognostic indicator for recurrence (p = 0.0032) and death (p = 0.0001), and was associated with poor response to both adjuvant (p = 0.0001) and palliative (p = 0.006) therapy. Analysis of the p53 gene with appropriate mutation detection methodology markedly improves the prediction of early recurrence, treatment failure, and death in breast cancer patients. Int. J. Cancer (Pred. Oncol.) 89:32–38, 2000.

Database of mutations in the p53 and APC tumor suppressor genes designed to facilitate molecular epidemiological analyses

Germline and somatic mutations in the p53 and APC genes contribute to neoplasia. The patterns of these and other acquired mutations in cancers reflect environmental mutagens and endogenous factors that contribute to carcinogenesis. Herein, we describe a database of almost 2,300 mutations in the p53 and APC genes published until September 1, 1993. In addition to cataloging the mutations, multiple fields of information have been added to facilitate future molecular epidemiological analyses of human cancer. The accuracy of the database has been checked by the present authors and, by soliciting feedback from the original corresponding authors. The strengths and limitations of the primary literature are discussed.

An evaluation of recombinant leukocyte A interferon with aspirin in patients with metastatic renal cell cancer

The authors designed a clinical trial to assess the impact of aspirin (ASA) (600 mg four times daily) on the constitutional sequelae of recombinant leakocyte A interferon (IFN‐α2A), 20 × 106 U/m2 thrice weekly, in 29 patients with advanced renal cell cancer (RCC). Aspirin provided no meaningful amelioration of side effects compared to our prior experience of IFN‐α2A alone. Interestingly, the objective response rate of 34% (10/29) was considerably higher than the 15% recently reported from an aggregate of 344 patients participating in 14 prospective clinical trials. In light of small numbers, subtle selection biases, and the well‐recognized hazards of retrospective analyses, currently it is unclear if the apparent therapeutic advantage from ASA plus IFN‐α2A reflects chance occurrence or therapeutic potentiation from ASA. A randomized trial is planned to determine if ASA may have enhanced the efficacy of IFN‐α2A in patients with advanced RCC.

Cell aggregates in the soft agar "human tumour stem-cell assay".

We evaluated colony formation in soft agar by cells obtained after mechanical and/or enzymatic disaggregation of 455 malignant human tumours. Counting and assessment of cell colonies in the agar plates were done by inverted microscopy, computerized image analysis, and inspection of serial photomicrographs of the agar plates. Our results indicate that standard methods of tumour disaggregation did not usually produce single-cell suspensions and that aggregates of tumour cells varying greatly in size were placed in the agar. Most groupings of cells identified as colonies 1-3 weeks after plating arose from enlargement of preexisting aggregates of cells.

Pharmacokinetics of mitomycin C in rabbit and human

SummaryA sensitive and specific high-pressure liquid chromatographic assay was developed to characterize the plasma elimination and urinary excretion of mitomycin C in humans. Extraction of mitomycin C and an internal standard, porfiromycin, from plasma by chromatography over a non-ionic resin, Porapak Q, yields high recovery of both compounds and facilitates measurement of as little as 5 ng mitomycin C by reversed-phase high-pressure liquid chromatography. The assay was used to characterize the plasma elimination of mitomycin C in rabbits and was shown to be applicable to the characterization of the pharmacokinetics of mitomycin C in humans receiving as little as 8 mg/m2.

High frequency of p53 gene mutations in primary breast cancers in Japanese women, a low-incidence population

The pattern of acquired mutations in the p53 tumour-suppressor gene is potentially useful for determining factors contributing to carcinogenesis in diverse populations differing in incidence and/or mortality from the disease. We previously reported differences in mutational patterns of the p53 gene in primary breast cancers from Midwest US Caucasian, African-American and Austrian women. Herein, we report 16 mutations in 27 primary breast cancers from Japanese women from Hirosaki, a population with a low incidence of breast cancer. The frequency of 59.3% of p53 mutations is the highest reported in breast cancers from a particular ethnic group thus far. A relatively high number of mutations (7/16) were heterozygous in at least some tumour cell clusters. Intergroup comparisons of the mutational pattern between this population and several other US, European and Japanese populations do not show any statistically significant differences. There were recurrent mutations at two sites, codon 273 (R --> H; three mutations), a common hotspot of mutations in breast and other cancers, and codon 183 (S --> Stop; two mutations), a very rare location for p53 mutations. These mutations were shown to be independent and presumably not in the germ line. The highest frequency of p53 mutations raises the possibility that p53 mutagenesis is a predominant factor for breast cancer development in this low-risk Japanese group, whereas in other cohorts different mechanisms are likely to account for the higher proportion of breast cancer. Further studies are needed to confirm the present observations.