Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro.

The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 +/- 0.5% in control cells to 7.0 +/- 0.3% in cells treated with 25 nM dDAVP (P less than 0.001), whereas cells doubling every 26 h showed an increase from 10.8 +/- 1.2% in control cells to 21.0 +/- 2.0% in cells treated with 150 nM dDAVP (P less than 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P less than 0.01) and of total [3H]diacylglycerols after incubation for both 3 min (P less than 0.05) and 9 min (P less than 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C.

Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia.

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal. The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells). By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines. The level of glutathione transferase activity was determined in whole cell extracts. Enzyme activity (mean +/- SE) in sensitive cells was 9.49 +/- 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 +/- 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 +/- 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01). Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells. Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells. Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant. Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells. Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement. Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells. Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines. Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells. These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Bayesian monitoring of phase II trials in cancer chemoprevention.

Early randomized Phase II cancer chemoprevention trials which assess short-term biological activity are critical to the decision process to advance to late Phase II/Phase III trials. We have adapted published Bayesian interim analysis methods (Spiegelhalter et al., J. R. Statist. Soc A, 1994; 157: 357-416) which give greater flexibility and simplicity of inference to the monitoring of randomized controlled Phase II trials using intermediate endpoints. The Bayesian stopping rule is designed to stop the trial more quickly when the evidence suggests ineffectiveness rather than when it suggests biological activity, thus allowing resources to be concentrated on those agents that show the most promise in this early stage of testing. We investigate frequentist performance characteristics of the proposed method through simulation of randomized placebo controlled trials with a growth factor intermediate end-point using mean and variance values derived from the literature. Simulation results show expected error rates and trial size similar to other commonly used group sequential methods for this setting. These results suggest that the Bayesian approach to interim analysis is well suited for monitoring small randomized controlled Phase II chemoprevention trials for early detection of either inactive or promising agents.

Models for early chemoprevention trials in breast cancer.

Several models are being explored for use in the phase I and phase II evaluation of breast cancer chemoprevention agents. The short-term DCIS/small invasive cancer model is probably best used in late phase I trials in conjunction with agents likely to have activity in the progression phase of neoplastic development in addition to activity in earlier phases. The core biopsy or FNA hyperplasia models may be best used with drugs that are likely to have activity primarily in the promotion phase of neoplastic development and that are suitable for longer duration trials lasting several months to years. Morphology currently is the key surrogate endpoint biomarker for assessing efficacy in phase II trials. Other biomarkers that may undergo modulation will have to be validated, in that modulation will have to be shown to be directly related to decreased cancer risk in subsequent phase III trials. Only then can they be considered as validated surrogate endpoint biomarkers and used as stand-alone efficacy markers in phase II trials. Despite accrual challenges and technologic hurdles, interest in phase I and phase II chemoprevention trials is high.

Concomitant chemotherapy and reirradiation as management for recurrent cancer of the head and neck.

Thirty-five patients with inoperable recurrent head and neck cancer previously treated with definitive irradiation were treated with reirradiation and concomitant chemotherapy. Patient records were retrospectively reviewed to assess toxicity, response, and survival. Patients received one of three regimens: 1) 40 Gy total (2 Gy daily), 300 mg/m2 5-fluorouracil intravenous bolus, and 2 g hydroxyurea orally daily for 5 days; 2) 48 Gy total (1.2 Gy twice daily), 300 mg/m2 5-fluorouracil intravenous bolus, and 1.5 g hydroxyurea orally daily for 5 days; 3) 60 Gy total (1.5 Gy twice daily), 300 mg/m2 5-fluorouracil intravenous bolus, and 1.5 g hydroxyurea orally daily for 5 days. For all regimens, treatment was given only on weeks 1, 3, 5, and 7. Acute toxicity was mainly hematologic and was less severe with the lower hydroxyurea dose. Acute mucosal and skin toxicity was acceptable for all regimens. Late toxicity was noted in 4 of 17 patients who survived 12 months or more. Late effects were Radiation Therapy Oncology Group grade 3 or less. Fifteen of 35 patients achieved a complete response, and 11 of 35 patients achieved a partial response. The median survival rate was 10.5 months. There was no significant difference in responses or median survival between the groups. Reirradiation of head and neck cancer with 5-fluorouracil and hydroxyurea offers acceptable acute toxicity and minimal late effects. The clinical response rates and median survival are encouraging. Further investigation is warranted.

Biomarkers for breast cancer.

Molecular biomarkers for breast cancer are of several types. Risk biomarkers are those associated with increased cancer risk and include mammographic abnormalities, proliferative breast disease with or without atypia, family clustering and inherited germ-line abnormalities. Surrogate endpoint biomarkers are tissue, cellular or molecular alterations that occur between cancer initiation and progression. These biomarkers are utilized as endpoints in short-term chemoprevention trials. Prognostic biomarkers provide information regarding outcome irrespective of therapy, while predictive biomarkers provide information regarding response to therapy. Candidate prognostic biomarkers for breast cancer include elevated proliferation indices such as Ki-67 and proliferating cell nuclear antigen (PCNA); ER and PR overexpression; markers of oncogene overexpression such as c-erbB-2, TGF-a and EGFr; indicators of apoptotic imbalance including overexpression of bcl-2 and an increased bax/bcl-2 ratio; markers of disordered cell signaling such as p53 nuclear protein accumulation; alteration of differentiation signals such as overexpression of c-myc and related proteins; loss of differentiation markers such as TGF-b II receptor and retinoic acid receptor; and alteration of angiogenesis proteins such as VEGF overexpression. As our knowledge regarding molecular biomarkers for breast cancer increases, prognostic indices will be developed that combine the predictive power of individual molecular biomarkers with specific clinical and pathologic factors.

An intron binding protein is required for transformation ability of p53.

Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.

Inhibition of the mutant p53 gene in transformation assays.

Mutation of the p53 gene is a key element in the development of several human cancers. Intron 4, a noncoding region of the p53 gene, is required for optimal expression of that gene. We have previously shown that nuclear protein binds intron 4 and have defined the protein-binding site. In this paper we address the question, "Does the mutant p53 gene's ability to transform cells to the malignant phenotype depend on protein binding to intron 4?" Using an in vitro assay in which the mutant p53 gene and Ha-ras oncogene cooperate in transformation of cells to the malignant phenotype, we determined the ability of mutant mouse p53 gene constructs, with and without two base pair substitutions at the intron 4 protein-binding site, to participate in malignant transformation. On Day 1, 5 x 10(5) rat embryo fibroblasts were transfected by the calcium phosphate procedure with 10 micrograms of both a mutant p53 gene construct and Ha-ras oncogene. Malignant transformation was evidenced by the formation of discrete foci of heaped-up cells. After 14 days of incubation at 37 degrees C in DMEM and 10% fetal calf serum (8% CO2), the cells were stained with cresyl violet and the foci counted. In three separate experiments, the presence of two base pair substitutions at the intron 4 protein-binding site caused a significant decrease in the number of foci formed (P less than 0.05).

Sentinel node biopsy for cutaneous melanoma in the head and neck.

BACKGROUND: Selective sentinel lymphadenectomy has gained widespread acceptance for staging of melanomas arising in the trunk and extremities, but the complex lymphatic drainage of the head and neck area has limited its application in this area. METHODS: We performed a retrospective analysis of patients who underwent selective sentinel lymphadenectomy for cutaneous melanoma of the head and neck at the University of Alabama at Birmingham from 1997 through 2000, by using a standard technique of preoperative lymphoscintigram and biopsy guided with blue dye injection and a handheld gamma probe. Complete lymph node dissection was recommended only for tumor-positive sentinel lymph nodes (SLNs). Survival curves were constructed with the Kaplan-Meier method. Fisher's exact test was used for comparisons. Significance was defined as P < .05. RESULTS: Thirty-eight patients underwent selective sentinel lymphadenectomy with the standard technique during the study period. A majority (82%) of patients were men with a median age of 55 years. The most common site of the primary tumor was the face (44%), followed by the scalp (24%). Mean tumor thickness was 2.5 mm. The sentinel node was identified during surgery in 35 patients (92%). Before the use of the handheld gamma probe, the identification rate of the SLN was only 56%. A single SLN was identified in 53% of cases. The incidence of metastases in SLN was 11.4%. With a mean follow-up of 17 months, the actuarial 3-year overall survival was 92%. The accuracy of the selective sentinel lymphadenectomy in this series was 80%. CONCLUSIONS: Selective sentinel lymphadenectomy in the head and neck region is a technically demanding procedure, but the combined use of blue dye and gamma-probe radiolocalization can be a reliable method of staging regional lymph nodes and determining the need for elective lymphadenectomy.

Multimodality treatment of Merkel cell carcinoma: case series and literature review of 1024 cases.

BACKGROUND: Merkel cell carcinoma (MCC) is an unusual and potentially aggressive cancer of the skin. There is no consensus regarding the optimal therapeutic approach, and the relative roles of surgery, radiotherapy, and chemotherapy still are controversial The aim of this study is to analyze the roles of these therapeutic options. METHODS: The medical records of 16 patients with a diagnosis of localized, primary MCC treated at the University of Alabama at Birmingham were reviewed. An extensive review of the English-language literature also was performed. The Kaplan-Meier method was used to develop the survival curves. Comparisons were made using Fisher's exact test. Significance was defined as P < .05. RESULTS: MCC presented primarily in Caucasians (98.3%) with a median age of 69 years. Immunosuppressive therapy appeared to play a role in the development of this cancer. In the UAB experience, 3-year actuarial survival was 31%. The only factor significantly associated with overall survival was the stage of disease at presentation: median survivals were 97 vs. 15 months for stages I and II, respectively (log-rank, P = .02). From the literature review, adjuvant radiotherapy was associated with a reduced risk of local recurrence (P < .00001). CONCLUSIONS: MCC is an aggressive cancer, with a high tendency for local recurrence and distant spread. Surgery and adjuvant radiotherapy appear to provide optimal local control. The role of chemotherapy remains to be defined.

CD44 expression in dysplastic epithelium and squamous-cell carcinoma of the esophagus.

The molecular events involved in the development of esophageal dysplasia and carcinoma are poorly understood. We examined the expression of CD44, a cell-adhesion molecule, in normal and dysplastic epithelia and in squamous-cell carcinoma (SCC) of the esophagus. A monoclonal antibody (MAb) which recognized all CD44 isoforms and 2 MAbs specific to the CD44v3 and CD44v6 splice variants were used to detect CD44 isoforms in 50 archival specimens. A semi-quantitative scoring system based on the extent and intensity of the immunostaining was used to quantify CD44 expression. In normal epithelium, expression of CD44 was strongest in the basal-cell layer and weak or absent in surface cells. Expression of CD44 was increased in dysplastic epithelium as compared with normal epithelium. The extent of this increase correlated directly with the severity of dysplasia. CD44 was expressed in all SCCs, but the extent and intensity of immunostaining varied with areas of tumor differentiation. The well-differentiated components showed greater CD44 expression than the moderately and poorly differentiated components. The patterns of expression of CD44v3 and CD44v6 were strikingly similar to that of total CD44 in normal, dysplastic and malignant esophageal epithelia. Thus, changes in expression of these splice variants likely account to some extent for the changes in total CD44 expression observed in the dysplastic and malignant transformation of the esophagus. Our results suggest that changes in the expression of CD44 may be involved in the development of esophageal dysplasia as well as SCC.

T1 and T2 squamous cell carcinoma of the oral tongue: prognostic factors and the role of elective lymph node dissection.

BACKGROUND: The management of micrometastatic disease from squamous cell carcinoma (SCC) of the oral tongue remains controversial. This study describes prognostic factors in the disease and reviews the role of elective neck dissection (END). METHODS: A retrospective analysis of all patients undergoing definitive surgical treatment of T1 and T2 SCC of the oral tongue between 1956 and 1994 at the University of Alabama at Birmingham was performed. RESULTS: Patient, disease, and treatment variables were compiled for 169 patients. Multivariate analysis showed age (p = .02), sex (p = .02), disease differentiation (p = .0003), and palpable lymphadenopathy (p = .02) to be significant prognostic variables. Fifteen patients underwent END and 6 were shown to have micrometastatic disease (40.0%). There were no neck recurrences in these patients, but END was not shown to improve survival. CONCLUSIONS: The presence of poorly differentiated disease gave the worst prognosis in this population of patients with T1 and T2 SCC of the oral tongue. A high incidence of nodal micrometastatic disease and the absence of recurrent disease after END suggest that END is appropriate therapy for these patients.

Retinoid modulation of biomarkers in oral leukoplakia/dysplasia.

Among the tissue, cellular, and molecular changes which take place during the development of squamous cell carcinoma (SCC) of the upper aerodigestive tract, only a limited number can be used as surrogate endpoint biomarkers (SEBs) in cancer chemoprevention trials. Molecular SEBs will be genes or gene products which can be measured accurately and reliably, are altered in intraepithelial neoplasia (dysplasia), correlate strongly with the true outcome (invasive cancer), and are modulated by a chemoprevention agent(s). To identify and modulate molecular SEBs in intraepithelial neoplasia of the upper aerodigestive tract, we studied expression of the epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha), and HER-2/neu genes in oral leukoplakia before, during, and after treatment with 13-cis-retinoic acid, a vitamin A derivative. Four of nine patients treated for 3 months with 1 mg/kg/day of 13-cis-retinoic acid had complete resolution of their leukoplakia. Biopsies were taken of leukoplakia and adjacent normal-appearing mucosa before, during, and after treatment. Immunohistochemistry was performed using the BioGenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system. Pretreatment expression of EGFR, TGF-alpha, and HER-2/neu in leukoplakia was increased when compared to normal-appearing mucosa. TGF-alpha expression decreased during treatment in leukoplakia, but not in normal-appearing mucosa, suggesting that TGF-alpha may serve as an intermediate endpoint in cancer chemoprevention trials.

Transforming growth factor alpha (TGF-alpha) expression in dysplastic oral leukoplakia: modulation by 13-cis retinoic acid.

BACKGROUND: Surrogate endpoint biomarkers (SEBs) are detectable molecular, cellular, and tissue changes that take place during tumorigenesis and can be modulated by a chemoprevention agent. METHOD: To identify candidate SEBs for invasive squamous cell carcinoma of the upper aerodigestive tract (SCC), we have studied the expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFr) in sequential biopsy specimens of dysplastic oral leukoplakia and adjacent normal-appearing mucosa. Biopsies were taken from patients before, during, and after treatment with 13-cis retinoic acid, a vitamin A derivative. Immunohistochemistry was performed using the Biogenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system. RESULTS: The pretreatment expression of TGF-alpha and EGFr in dysplastic oral leukoplakia was increased when compared with their expression in adjacent normal-appearing mucosa (p = 0.001 and p = 0.01, respectively). Eleven of 14 patients enrolled in the study (78.6%) completed 3 months of treatment with 13-cis retinoic acid (1. 0 mg/kg/day). TGF-alpha expression in dysplastic oral leukoplakia, but not in adjacent normal-appearing mucosa, decreased during treatment (p < 0.05). CONCLUSIONS: TGF-alpha is a candidate SEB for future SCC chemoprevention trials.

Prognostic factors in minor salivary gland cancer.

BACKGROUND: Minor salivary gland cancer occurs infrequently and presents a diagnostic and therapeutic challenge. The purpose of this study was to determine prognostic factors for this disease. METHODS: The medical records of 95 patients diagnosed and treated at the University of Alabama at Birmingham over a 35-year period were reviewed. Information concerning patient, disease, and treatment characteristics was compiled for each case. Multivariate analysis was conducted using a rank regression procedure. RESULTS: State I or II cancer (p = .022), the absence of cervical lymph node metastases (p = .001), and surgical margins which were free of cancer (p < .001) were predictive of increased 4-year disease-free survival by multivariate analysis. CONCLUSION: Our findings emphasize the need for detection of early-stage disease combined with complete surgical extirpation of the cancer, which provide the patient with the best chance for locoregional control and long-term survival.

Molecular biomarkers for breast cancer prognosis: coexpression of c-erbB-2 and p53.

OBJECTIVE: To assess the prognostic significance of molecular biomarkers, particularly c-erbB-2 and p53, through study of prospective clinical data and archival breast cancer tissues for women accrued to the Alabama Breast Cancer Project. SUMMARY BACKGROUND DATA: Defining molecular abnormalities in breast cancer is an important strategy for early detection, assessment of prognosis, and treatment selection. Evidence is strong that selective biomarkers, including c-erbB-2 and p53, have prognostic significance in breast cancer. Few studies have analyzed the prognostic significance of coexpression of biomarkers. METHODS: Study patients were those accrued to the Alabama Breast Cancer Project (1975-1978) who had archival breast cancer tissues available for analysis. Criteria for entrance into the Alabama Breast Cancer Project were T1-3 breast cancer with M0 status. Age, nodal status, and histologic grade were also documented. Patients were randomized to radical versus modified radical mastectomy, and node-positive patients were also randomized to adjuvant chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil [CMF]) versus melphalan. Archival breast cancer tissues were studied for c-erbB-2, TGF-alpha, p53, cathepsin D, bcl-2, and estrogen and progesterone receptor expression using immunohistochemistry. Survival curves were developed using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test, multivariate analysis using a rank regression model. RESULTS: Three hundred eleven patients were accrued to the Alabama Breast Cancer Project, and paraffin-embedded breast cancer tissues for 90 patients were available for immunohistochemical analysis of molecular biomarkers. Univariate analysis showed nodal status, c-erbB-2 expression, and p53 expression to have prognostic significance. Coexpression of c-erbB-2 and p53 was also found to have prognostic significance by the log-rank test. Multivariate analysis showed T stage, nodal status, c-erbB-2 expression, and p53 expression to have independent prognostic significance. CONCLUSIONS: These data suggest that c-erbB-2 and p53 expression in breast cancer have prognostic significance. After median follow-up of 16 years, coexpression of c-erbB-2 and p53 may have more prognostic significance than traditional prognostic factors such as T stage and nodal status. Prospective study of large numbers of patients with breast cancer is encouraged to validate these findings.