National Outcomes of Liver Transplantation for Model for End-Stage Liver Disease Score ≥40: The Impact of Share 35.

In certain regions of the United States in which organ donor shortages are persistent and competition is high, recipients wait longer and are critically ill with Model for End-Stage Liver Disease (MELD) scores ≥40 when they undergo liver transplantation. Recent implementation of Share 35 has increased the percentage of recipients transplanted at these higher MELD scores. The purpose of our study was to examine national data of liver transplant recipients with MELD scores ≥40 and to identify risk factors that affect graft and recipient survival. During the 12-year study period, 5002 adult recipients underwent deceased donor whole-liver transplantation. The 1-, 3-, 5- and 10-year graft survival rates were 77%, 69%, 64% and 50%, respectively. The 1-, 3-, 5- and 10-year patient survival rates were 80%, 72%, 67% and 53%, respectively. Multivariable analysis identified previous transplant, ventilator dependence, diabetes, hepatitis C virus, age >60 years and prolonged hospitalization prior to transplant as recipient factors increasing the risk of graft failure and death. Donor age >30 years was associated with an incrementally increased risk of graft failure and death. Recipients after implementation of Share 35 had shorter waiting times and higher graft and patient survival compared with pre-Share 35 recipients, demonstrating that some risk factors can be mitigated by policy changes that increase organ accessibility.

GSTPi-positive tumour microenvironment-associated fibroblasts are significantly associated with GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and axillary lymph node metastases.

BACKGROUND: Glutathione S-transferase Pi (GSTPi) expression is one of the factors, which is known to be associated with development of resistance to chemotherapeutics in cancer patients, including those with breast cancer. Yet, its expression has been reported to be undetectable in cancer cells in high percent of patients with primary breast cancer. However, GSTPi expression in stromal cells in breast tumour microenvironment, namely cancer-associated fibroblast (CAF), which is recognised to have major roles in cancer progression, remains poorly reported. METHODS: The aim of the study was to determine the expression of GSTPi; vimetin, a fibroblast-associated cytoskeleton protein; and α-smooth muscle actin (α-SMA), a known marker of CAF in breast cancer tissue, by immunohistochemical staining method in consecutive histologic sections of formalin-fixed and paraffin-embedded tissue biopsy specimens from a cohort of 39 paired cases of patients with invasive breast cancer and the corresponding axillary lymph nodes metastases. RESULTS: Ductal and acinar luminal epithelial cells, myoepithelial cells and surrounding fibroblasts exhibited a homogeneous cytoplasmic reactivity with anti-GSTPi antibody in 11 of 11 cases of benign breast tissue biopsies. The vimentin-positive fibroblasts were unreactive with anti-α-SMA antibody. Loss of GSTPi expression was observed in breast cancer cells, at both the primary and metastatic sites, in 31 of 39 paired cases, as compared with benign breast epithelial cells (Fisher's exact test P<0.001). A significant association was observed between GSTPi-positive, vimentin-positive and α-SMA-positive fibroblast in tumour microenvironment at both sites. CONCLUSION: This is an original report of demonstration of a significance association between tumour microenvironment-associated GSTPi-positive CAF (vimentin/α-SMA-positive) and the GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and the corresponding axillary lymph nodes metastases.

Genetic variations in angiogenesis pathway genes associated with clinical outcome in localized gastric adenocarcinoma.

BACKGROUND: Angiogenesis has been attributed to be a well-recognized aspect of human cancer biology. As such, proteinase-activated receptor (PAR)-1, endostatin (ES) and interleukin-8 (IL-8) mediate the regulation of early-onset angiogenesis and in turn impact the process of tumor-growth and disease progression. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tissues were obtained from 137 patients with localized gastric cancer at University of Southern California and Memorial Sloan-Kettering Cancer Center medical facilities. DNA was extracted and genotyping was carried out using PCR-restriction fragment length polymorphism-based protocols. RESULTS: In false discovery rate-adjusted univariate analysis, PAR-1 -506 ins/del (P < 0.001), ES +4349 G>A (P = 0.004), and IL-8 -251 T>A (P < 0.0001) were associated with time to tumor recurrence (TTR). Further, PAR-1 -506 ins/del and IL-8 -251 were associated with overall survival (OS). After adjusting for covariates, IL-8 remained significantly associated with TTR (adjusted P = 0.003) and OS (adjusted P = 0.049), whereas ES was significantly associated with TTR (adjusted P = 0.026). CONCLUSIONS: Polymorphisms in PAR-1, ES, and IL-8 may serve as independent molecular prognostic markers in patients with localized gastric adenocarcinoma. The assessment of the patients' individual risk on the basis of interindividual genotypes may therefore help to identify patient subgroups at high risk for poor clinical outcome.

Urinary tract infections and reduced risk of bladder cancer in Los Angeles.

We investigated the association between urinary tract infections (UTIs) and transitional cell carcinoma of the bladder in a population-based case-control study in Los Angeles covering 1586 cases and age-, gender-, and race-matched neighbourhood controls. A history of bladder infection was associated with a reduced risk of bladder cancer among women (odds ratio (OR), 0.66; 95% confidence interval (CI), 0.46-0.96). No effect was found in men, perhaps due to power limitations. A greater reduction in bladder cancer risk was observed among women with multiple infections (OR, 0.37; 95% CI, 0.18-0.78). Exclusion of subjects with a history of diabetes, kidney or bladder stones did not change the inverse association. A history of kidney infections was not associated with bladder cancer risk, but there was a weak association between a history of other UTIs and slightly increased risk among men. Our results suggest that a history of bladder infection is associated with a reduced risk of bladder cancer among women. Cytotoxicity from antibiotics commonly used to treat bladder infections is proposed as one possible explanation.

Molecular profiling including epidermal growth factor receptor and p21 expression in high-risk breast cancer patients as indicators of outcome.

BACKGROUND: Patients with high-risk primary breast cancer remain at high risk for relapse. More precise prognostic and predictive tools are needed to improve treatment of such patients. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tumors from 239 high-risk breast cancer patients were examined for expression of human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), estrogen receptor, progesterone receptor, Ki-67, p16, p21, p27, and p53 by immunohistochemistry. Gene expression of EGFR, HER2, glutathione S-transferase-Pi (GSTP1), excision repair cross complementation1 (ERCC1), p21, beta-tubulin-3, multidurg resistance (MDR1), cyclooxygenase2 (COX2), and cyclin-E was measured by RT-PCR. RESULTS: Eighty percent of patients presented with locally advanced, or > or =10 axillary nodal metastasis, and 20% with inflammatory breast cancer. The median age was 46 years (26-62 years) and the median number of involved axillary lymph nodes was 12 (0-42). At a median follow-up of 86 months, relapse-free survival (RFS) and overall survival for the entire group were 50% (95% CI 43% to 57%) and 62% (95% CI 56% to 69%). Multivariate Cox stepwise analysis resulted in a simple model for RFS consisting only of p21 expression, EGFR expression assessed by RT-PCR, and number of axillary nodal metastases. CONCLUSION: A prognostic model on the basis of the expression of a limited number of proteins and genes may help to guide target-specific therapies in patients with high-risk breast cancer.

Polymorphisms in VEGF and IL-8 predict tumor recurrence in stage III colon cancer.

BACKGROUND: Identifying molecular markers for tumor recurrence is critical in successfully selecting patients with stage III colon cancer who are more likely to benefit from adjuvant chemotherapy. The present study analyzed a subset of 10 polymorphisms within eight genes involved in the tumor angiogenesis pathway and their impact on prognosis in stage III colon cancer patients treated with adjuvant chemotherapy. PATIENTS AND METHODS: Blood samples were obtained from 125 patients with locally advanced colon cancer at University of Southern California medical facilities. DNA was extracted from peripheral blood and the genotypes were analyzed using PCR-restriction fragment length polymorphism and 5'-end [gamma-(33)P] ATP-labeled PCR protocols. RESULTS: Polymorphisms in vascular endothelial growth factor (VEGF) (C+936T; P = 0.003, log-rank test) and interleukin-8 (IL-8) (T-251A; P = 0.04, log-rank test) were independently associated with risk of recurrence in stage III colon cancer patients. In combined analysis, grouping alleles into favorable versus nonfavorable alleles, high expression variants of VEGF C+936T and IL-8 T-251A were associated with a higher likelihood of developing tumor recurrence (P < 0.001). CONCLUSION: High expression variants of VEGF C+936T and IL-8 T-251A were associated with shorter time to tumor recurrence, indicating that the analysis of angiogenesis-related gene polymorphisms may help to identify patient subgroups at high risk for tumor recurrence.

Prolonged continuous infusion of fluorouracil with weekly bolus leucovorin: a phase II study in patients with disseminated colorectal cancer.

BACKGROUND: Biochemical modulation of bolus fluorouracil (5-FU) by addition of leucovorin to the treatment regimen has increased response in patients with disseminated colorectal cancer from fewer than 20% to more than 40%. In view of the short half-life of 5-FU and its cell cycle specificity, it may be that infusion rather than intravenous bolus injection would increase efficacy. Furthermore, the advent of safer indwelling intravenous catheters and pump technology, allowing home and ambulatory treatment, has made protracted infusion clinically feasible. To examine these questions, we conducted a phase I trial using protracted infusion of 5-FU by indwelling catheter and pump, with leucovorin given by bolus injection, and reported 40% partial response. PURPOSE: We have now initiated a phase II study of 5-FU given by prolonged continuous infusion with weekly bolus injections of leucovorin in previously untreated patients with measurable, disseminated colorectal cancer. METHODS: Forty-one patients were treated. The regimen consisted of treatment for 4 weeks with 5-FU at a dose of 200 mg/m2 daily as a continuous infusion by indwelling intravenous catheter and pump, followed by a 2-week rest and then by monthly cycles of 3 weeks of treatment and 1-week rest until disease progression. Leucovorin was given as a bolus injection of 20 mg/m2 at the beginning of each week of treatment with 5-FU. RESULTS: Nineteen (46%) of 41 patients had objective response: Three complete responses and 16 partial responses were seen. Overall, the median duration of response was 8 months. The median duration of survival was 16 months: 18 months for responders and 10 months for nonresponders. In general, toxic effects were mild and consisted primarily of stomatitis and palmar-plantar erythrodysesthesia (hand-foot syndrome). Neither grade 4 toxic effects nor treatment-related deaths were observed. The only serious side effects were catheter thrombosis (three patients) and catheter sepsis (one patient). CONCLUSION: We conclude that this safe regimen is one of the most effective for the treatment of disseminated colorectal cancer. IMPLICATIONS: The regimen should be tested prospectively against other regimens in use for this disease. It is currently included in a phase III study of the Southwest Oncology Group for this purpose. That study will assess quality of life as well as response rates and survival duration.

Association of p27Kip1 levels with recurrence and survival in patients with stage C prostate carcinoma.

BACKGROUND: There are few biologic determinants that are prognostic for patients with localized prostate cancer. We examined whether cellular levels of the cyclin-kinase inhibitor p27Kip1 (also known as p27) in prostate tumors could be used to predict progression of this disease. METHODS: Levels of p27 in tumor cell nuclei were assessed by immunohistochemical analysis of tissue sections from the primary tumors of 96 patients with stage C prostate carcinoma who had been treated by radical prostatectomy. Tumors were classified into one of the following three groups on the basis of the percentage of tumor cells showing nuclear p27 reactivity: low (0%-10%), moderate (11%-50%), and high (>50%). The Mantel-Haenszel test, Kaplan-Meier analysis, and the logrank test were used to calculate the probability that nuclear p27 levels were associated with tumor grade and substage, with a serum prostate-specific antigen (PSA) recurrence (defined as the finding of a detectable level [0.4 ng/mL or greater] of serum PSA following radical prostatectomy), with the recurrence of clinically evident disease, and with survival. All reported P values are two-sided. RESULTS: Luminal cells and basal cells of normal prostate glands showed high levels of nuclear p27 immunoreactivity in all tissue sections examined. Fifty-three tumors showed high p27 reactivity, 31 showed moderate reactivity, and 12 showed low or no detectable reactivity. Decreased levels of p27 were associated with tumor grade (P = .004). Tumor levels of p27 were not associated with preoperative prostate-specific antigen levels (P = .360) or with tumor substage (P = .320). However, decreased p27 reactivity was significantly associated with an increased probability of recurrence (P = .004) and decreased survival (P = .010). The median recurrence-free interval for patients with tumors showing high, moderate, or low p27 reactivity was 13.7 years, 8.4 years, and 4.7 years, respectively. Median survival times were more than 14 years, more than 13.5 years, and 8.1 years for patients in the high, moderate, and low p27 reactivity groups, respectively. CONCLUSION: Levels of nuclear p27 immunoreactivity in the primary tumor can be used to predict recurrence and survival among patients with localized prostate cancer.

Susceptibility of nonpromoter CpG islands to de novo methylation in normal and neoplastic cells.

BACKGROUND: Many cancers display alterations in methylation patterns of CpG islands--stretches of DNA rich in CpG dinucleotides often associated with gene promoters that are involved in initiation of gene transcription. This methylation may perturb expression of genes critical to the regulation of cell proliferation. Aberrant methylation is not limited to a few genes or to promoter regions but has been found on a genome-wide scale in a variety of neoplasias, including colorectal cancer and acute myelogenous leukemia. Our goal was to characterize, in a quantitative manner, the profiles of abnormally methylated genes that may be specific for different cancers. METHODS: Using a quantitative assay, methylation-sensitive single nucleotide primer extension (MS-SNuPE), we have analyzed the methylation levels of promoter and exonic (coding region) CpG islands of two cyclin-dependent kinase inhibitors [p15(INK4B) and p16(INK4A)] and the PAX6 gene, which encodes a transcriptional factor involved in neuronal proliferation, in DNA samples taken from patients with chronic myelogenous leukemia, acute myelogenous leukemia, myelodysplastic syndrome, and colorectal cancer. RESULTS: De novo methylation of all three exonic loci in tumors--relative to baseline levels found in nontumor tissue or blood--was observed in hematologic neoplasias and in solid tumors as well as in normal colonic tissue. However, methylation of promoter regions was more limited. Moreover, two different patterns of promoter methylation distinguished the leukemias from colorectal cancer: p15 promoter hypermethylation was found only in the leukemias, and p16 promoter hypermethylation occurred only in colon tumors. However, we did not address this issue prospectively; therefore, such an observation is only hypothesis generating. CONCLUSIONS: The methylation patterns that we observed suggest that exonic CpG islands are more susceptible to de novo methylation than promoter islands and that methylation may be seeded in exonic regions, from which it can spread to other islands, including promoter regions. Subsequent selection of cells with a growth advantage conferred by spread of methylation into and inactivation of a particular promoter might then contribute to the genesis of a specific type of cancer.

Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis.

BACKGROUND: Tumor stage, histologic grade, and regional lymph node status are currently used to obtain prognostic information about bladder cancers. However, additional prognostic indicators are needed to aid clinicians in selecting patients who would benefit most from specific therapies. A majority of studies assessing the prognostic value of measuring tumor angiogenesis (i.e., measurement of tumor microvessel densities) have found a positive association between increasing microvessel densities and worsening prognosis. PURPOSE: We explored the relationship between established prognostic indicators and the extent of tumor-associated angiogenesis in patients with invasive transitional cell carcinoma (TCC) of the bladder, and we determined whether tumor microvessel density measurement could be used independently to predict bladder tumor behavior. METHODS: Tumor tissue was obtained from 164 patients with invasive primary TCC of the bladder. The extent of tumor-associated angiogenesis in this tissue was evaluated by immunohistochemical methods using HPCA-1, a mouse monoclonal antibody directed against the endothelial cell antigen, CD34. The number of microvessels in a 200x microscopic high-power field (hpf) containing the area of greatest neovascularization within or immediately adjacent to each tumor was determined. The patient population was then divided into three equivalently sized groups, with tumors containing low (< or = 64), intermediate (65-99), or high (> or = 100) numbers of microvessels per hpf. Kaplan-Meier product limit estimates of overall survival and the complement of cumulative incidence curves for recurrence-free survival were plotted. When analyzing survival or recurrence, the logrank test was used to compare groups of patients with and without stratification according to tumor stage. Analysis of variance was used to test for an association between microvessel density and established prognostic variables. Reported P values are from two-sided tests. RESULTS: Microvessel density was significantly associated with disease-free (P < .0001) and overall (P = .0007) survival. The estimated probabilities of recurrence at 5 years were 19% (95% confidence interval [CI] = 8-29), 56% (95% CI = 43-69), and 68% (95% CI = 55-81) for patients with lowest, intermediate, and highest microvessel counts, respectively. Overall survival at 5 years was estimated to be 68% (95% CI = 56-81), 44% (95% CI = 30-57), and 34% (95% CI = 21-47) for the same three patient groups. Microvessel density was associated with disease progression in patients with organ-confined tumors, tumors extending through the bladder wall, and tumors that had spread to regional lymph nodes. Tumor angiogenesis was found to be an independent prognostic indicator when evaluated in the presence of histologic grade, pathologic stage, and regional lymph node status. CONCLUSION: Tumor angiogenesis, as determined by microvessel density measurement, is an independent prognostic indicator for patients with invasive TCC of the bladder.

Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression.

BACKGROUND: Thrombospondin-1 (TSP) is a 430-kd glycoprotein that is an important component of the extracellular matrix and is known to be a potent inhibitor of angiogenesis (i.e., formation of new blood vessels) both in vitro and in vivo. Several reports suggest that TSP possesses tumor suppressor function, possibly through its ability to inhibit tumor neovascularization. It has recently been shown that TSP expression is enhanced by the product of the p53 gene (also known as TP53). PURPOSE: We examined the role of TSP expression in tumor recurrence and overall survival in patients with invasive bladder cancer. We also examined the relationship between alterations in p53 protein expression, TSP expression, and tumor angiogenesis. METHODS: Tumors from 163 patients (with a median follow-up of 7.7 years) who underwent radical cystectomy for invasive transitional cell carcinoma of the bladder (63 patients with organ-confined disease and no lymph node involvement, 48 patients with extravesical extension of the disease and no lymph node involvement, and 52 patients with metastasis to regional lymph nodes) were examined for TSP expression by immunohistochemistry, utilizing monoclonal antibody MA-II, which recognizes an epitope in the amino-terminal region of TSP. For each tumor, microvessel density counts and p53 protein expression status (via immunohistochemistry) were also determined. TSP expression was graded as low, moderate, or high without knowledge of clinical outcome, p53 status, and microvessel density count; tumors with moderate and high TSP levels were considered as one group. Groups of patients were compared by Kaplan-Meier product limit estimates of overall survival, the complement of cumulative incidence curves for recurrence-free survival, and the stratified logrank test. Reported P values are two-sided. RESULTS: TSP expression was significantly associated with disease recurrence (P = .009) and overall survival (P = .023). Patients with low TSP expression exhibited increased recurrence rates and decreased overall survival. TSP expression was an independent predictor of disease recurrence (P = .002) and overall survival (P = .01) after stratifying for tumor stage, lymph node status, and histologic grade, but it was not independent of p53 status. TSP expression was significantly associated with p53 expression status (P = .001) and microvessel density counts (P = .001). Tumors with p53 alterations were significantly more likely to demonstrate low TSP expression, and tumors with low TSP expression were significantly more likely to demonstrate high microvessel density counts. Results of an analysis of variance were compatible with the hypothesis that p53 affects tumor angiogenesis by regulating the level of TSP expression. CONCLUSIONS AND IMPLICATIONS: These data support the concept that TSP may possess a tumor-inhibitory function. TSP may act, in part, through the regulation of tumor neovascularity. These results may also provide insight into one mechanism by which p53 exerts its tumor suppressor effects, i.e., through the control of tumor angiogenesis.

Effect of p21WAF1/CIP1 expression on tumor progression in bladder cancer.

BACKGROUND: Altered expression of p53 protein is an important predictor of progression in bladder cancer. The action of p53 on cell cycle regulation is mediated, in part, through expression of the cyclin-dependent kinase inhibitor p21WAF/CIP1 (p21). Loss of p21 expression may, therefore, contribute to tumor progression. We sought to determine the relationship between p21 expression in bladder cancer and disease progression. METHODS: Tumor specimens were obtained from 242 patients who underwent cystectomy for bladder cancer. Median follow-up was 8.5 years (range, 0.1-11.8 years). Nuclear p21 status was determined by immunohistochemistry and was then analyzed in relationship to the probability of tumor recurrence, overall survival, and tumor p53 status. Reported P values are two-sided. RESULTS: Nuclear p21 expression was detected in the tumors of 156 (64%) of the 242 patients. Patients with p21-positive tumors had a decreased probability of tumor recurrence (P<.00001) and an increased probability of overall survival (P<.00001) in comparison with patients with p21-negative tumors. In a multivariable analysis, p21 expression was an independent predictor of tumor recurrence (P = .0017) and of survival (P = .006) when assessed with tumor grade, tumor stage, lymph node status, and p53 status. p21 expression was associated with p53 status (P<.001); 56% of tumors with p53 alterations showed loss of p21 expression, whereas 79% of tumors expressing wild-type p53 were p21 positive. Patients with p53-altered/p21-negative tumors demonstrated a higher rate of recurrence and worse survival compared with those with p53-altered/p21-positive tumors (P<.0001). Patients with 53-altered/p21-positive tumors demonstrated a similar rate of recurrence and survival as those with p53-wild type tumors. CONCLUSION: Loss of p21 expression is a statistically significant and independent predictor of bladder cancer progression. Maintenance of p21 expression appears to abrogate the deleterious effects of p53 alterations on bladder cancer progression.

Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma.

BACKGROUND: The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. METHODS: We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P: value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. RESULTS: Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P =.016). CONCLUSION: The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.

Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy.

To determine whether neuroblastomas acquire a sustained drug-resistant phenotype from exposure to chemotherapeutic agents given to patients in vivo, we studied neuroblastoma cell lines established at different points of therapy: six at diagnosis before therapy (DX), six at progressive disease during induction therapy (PD-Ind), and five at relapse after intensive chemoradiotherapy and bone marrow transplantation (PD-BMT). Cells were maintained in the absence of drug selective pressure. Dose-response curves of melphalan, cisplatin, carboplatin, doxorubicin, and etoposide for the cell line panel were determined by measuring cytotoxicity with a 96-well-plate digital imaging microscopy (DIMSCAN) microassay. Drug resistance of cell lines progressively increased with the intensity of therapy delivered in vivo. The greatest resistance was seen in PD-BMT cell lines: IC90 values in PD-BMT cell lines were higher than clinically achievable drug levels by 1-37 times for melphalan, 1-9 times for carboplatin, 25-78 times for cisplatin, 6-719 times for doxorubicin, and 3-52 times for etoposide. Genomic amplification of MYCN did not correlate with resistance. Cross-resistance by Pearson correlation (r > or = 0.6) was observed between: (a) cisplatin + doxorubicin; (b) carboplatin + cisplatin, etoposide, or melphalan; (c) etoposide + cisplatin, melphalan, or doxorubicin. These data indicate that during therapy, neuroblastomas can acquire resistance to cytotoxic drugs because of the population expansion of tumor cells possessing stable genetic or epigenetic alterations that confer resistance.

A Xeroderma pigmentosum group D gene polymorphism predicts clinical outcome to platinum-based chemotherapy in patients with advanced colorectal cancer.

The Xeroderma pigmentosum group D (XPD) protein is an essential participant in nucleotide excision repair and basal transcription. There is evidence that three common polymorphisms of the XPD gene (C156A, Asp312Asn, and Lys751Gln) may be associated with differential DNA repair activity. Because increased DNA repair plays an important role in chemoresistance to platinum-based compounds, we assessed the aforementioned polymorphisms in 73 patients with metastatic colorectal cancer and determined their outcome to 5-fluorouracil/oxaliplatin. Among those tested for the Lys751Gln polymorphism, 24% (5 of 21) patients with the Lys/Lys genotype responded, versus 10% (4 of 39) and 10% (1 of 10) of those with the Lys/Gln and Gln/Gln genotypes (P = 0.015). The median survival for those with the Lys/Lys genotype was 17.4 (95% CI 7.9, 26.5) versus 12.8 (95% CI 8.5, 25.9) and 3.3 (95% CI 1.4, 6.5) months for patients with the Lys/Gln and Gln/Gln respectively (P = 0.002). The polymorphisms C156A and Asp312Asn of the XPD gene were not associated with response to 5-fluorouracil/oxaliplatin nor with survival. However, a linkage was observed between the Lys751 allele and the C156 allele (P = 0.028), and between the Lys751Lys genotype and the Asp312Asp genotype (P < 0.001). We conclude that XPD Lys751Gln polymorphism may be an important marker in the prediction of clinical outcome to platinum-based chemotherapy.

Induction of graft versus leukemia effect in bone marrow transplantation: dosage and time schedule dependency of interleukin 2 therapy.

The present work is a continuation of our studies to improve the graft versus leukemia (GVL) effect in autologous bone marrow transplantation. We have recently shown that the GVL effect of bone marrow transplantation (BMT) with interleukin 2 (IL-2)-activated bone marrow (ABM) followed by IL-2 therapy immediately after BMT is superior to the GVL effect of BMT with fresh, syngeneic bone marrow, with or without IL-2 therapy, in mice with acute myeloid leukemia. The present studies show that institution of IL-2 treatment 1, 2, or 3 weeks after BMT with ABM resulted in shortening of survival and fall in cure rate as compared to IL-2 therapy instituted immediately after BMT with ABM. Increasing the dose of IL-2 did not improve results. However, reducing the frequency of IL-2 administration to once a day instead of twice a day affected the results adversely. Commencing IL-2 therapy 1, 2, or 3 weeks after BMT with fresh, syngeneic bone marrow did not improve the GVL effect as compared to IL-2 therapy started immediately after BMT with fresh, syngeneic bone marrow. Cryopreserved bone marrow was effectively activated with IL-2 and used successfully for BMT after thawing. The animals cured of leukemia by BMT with ABM and and IL-2 therapy were not resistant to leukemia and died when reinfused with leukemic cells. Our findings suggest that for optimum GVL effect, activation of bone marrow is necessary and IL-2 therapy should be started immediately after BMT with ABM.

Inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin G1 construct.

Tumor invasion and associated angiogenesis evoke a remodeling of extracellular matrix components. Retroviral vectors bearing auxiliary matrix-targeting motifs (ie., collagen-binding polypeptides) accumulate at sites of newly exposed collagen, thus promoting tumor site-specific gene delivery. In this study, we assessed the antitumor effects of serial portal vein infusions of matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in a nude mouse model of liver metastasis. The size of tumor foci was dramatically reduced in dnG1 vector-treated mice compared with that in control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in the development of targeted injectable vectors for metastatic cancer.

dUTP nucleotidohydrolase isoform expression in normal and neoplastic tissues: association with survival and response to 5-fluorouracil in colorectal cancer.

Aberrant dUTP metabolism plays a significant role in the underlying molecular mechanisms of cell killing mediated by inhibitors of thymidylate biosynthesis. dUTP nucleotidohydrolase (dUTPase) is the key regulator of dUTP pools, and significant evidence exists suggesting that the expression of this enzyme may be an important determinant of cytotoxicity mediated by inhibitors of thymidylate synthase (TS). In this study, we have determined the expression patterns of dUTPase in normal and neoplastic tissues and examined the association between dUTPase expression and response to 5-fluorouracil (5-FU)-based chemotherapy and overall survival in colorectal cancer. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using a monoclonal antibody (MAb), DUT415, that cross-reacts with both nuclear and mitochondrial isoforms of human dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear dUTPase staining was observed exclusively in replicating cell types. This observation is in agreement with cell culture studies where expression of the nuclear isoform (DUT-N) is proliferation dependent In contrast, cytoplasmic expression of dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria. Consistent with this observation, cell culture studies reveal that the mitochondrial isoform (DUT-M) is expressed constitutively, independent of cell cycle status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N isoform, whereas cytoplasmic staining represents the DUT-M isoform. In colon cancer tumor specimens, expression of dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns of dUTPase protein expression observed included exclusive nuclear, exclusive cytoplasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistochemical detection of dUTPase in colon cancers provides distinct intracellular phenotypes of expression that may be of significant prognostic value. To examine the association between dUTPase expression and response to 5-FU-based chemotherapy and overall survival, we initiated a retrospective study including tumor specimens from 20 patients who had received protracted infusion of 5-FU and leucovorin for treatment of metastatic colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear dUTPase expression, 6 responded to 5-FU-based chemotherapy, 4 had stable disease, and 2 had progressive disease. Of the patients presenting positive nuclear dUTPase expression, 0 responded to chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005). The median survival for patients with tumors lacking nuclear staining was 8.5 months and 6.9 months for patients with tumors demonstrating positive nuclear dUTPase expression (P = 0.09). Time to progression was significantly longer for patients with tumors lacking nuclear staining (P = 0.017). Variable cytoplasmic dUTPase expression was observed in these tumors; however, there was no apparent association with clinical response or survival in this limited study. Nuclear dUTPase staining within these tumors was also associated with TS gene expression (P = 0.06). This study demonstrates that low intratumoral levels of nuclear dUTPase protein expression is associated with response to 5-FU-based chemotherapy, greater time to progression, and greater overall survival in colorectal cancer. Conversely, high levels of nuclear dUTPase protein expression predict for tumor resistance to chemotherapy, shorter time to progression, and shorter overall survival. This report represents the first clinical study implicating dUTPase overexpression as a mechanism of resistance to TS inhibitor-based chemotherapy.

Progressive increases in de novo methylation of CpG islands in bladder cancer.

We conducted a quantitative analysis of the extent of de novo methylation of four CpG islands in human urinary transitional cell carcinomas of different stages and grades to determine how frequently these CpG islands became methylated in transition cell carcinomas during progression. The CpG islands included exon 5 of PAX6, exon 2 of p16, the 5' end of the deleted in bladder cancer gene, and the 5' end of transmembrane protein containing epidermal growth factor and follistatin domains. These sequences were not methylated in normal urothelial tissues; however, 48 of the 54 tumors examined (89%) showed methylation levels in excess of 20% for at least one of the markers. The number of markers concurrently methylated in individual tumors increased with the stage of the tumor, with several of the more aggressive invasive cancers showing hypermethylation of all four markers compared with the less aggressive invasive cancers. However, considerable methylation defects were present in superficial, preinvasive, papillary tumors. These data demonstrate that 89% of bladder cancers have increased methylation of CpG islands relative to their normal counterparts and suggest the occurrence of a hypermethylator phenotype in which multiple independent CpG islands become concurrently methylated in individual tumors in a process associated with tumor progression.