Alan R. Schned

Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer

We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 × 10−10 and the allelic odds ratio was 1.15 (95% confidence interval 1.10–1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.

Incidence of transitional cell carcinoma of the bladder and arsenic exposure in New Hampshire

AbstractObjective: Arsenic is a known bladder carcinogen and populations exposed to high arsenic levels in their water supply have reported elevated bladder cancer mortality and incidence rates. To examine the effects of lower levels of arsenic exposure on bladder cancer incidence, we conducted a case–control study in New Hampshire, USA where levels above 10 μ/l are commonly found in private wells. Methods: We studied 383 cases of transitional cell carcinoma of the bladder cancer, newly diagnosed between July 1, 1994 and June 30, 1998 and 641 general population controls. Individual exposure to arsenic was determined in toenail clippings using instrumental neutron activation analysis. Results: Among smokers, an elevated odds ratio (OR) for bladder cancer was observed for the uppermost category of arsenic (OR: 2.17, 95% CI: 0.92–5.11 for greater than 0.330 mcg/g compared to less than 0.06 μ/g). Among never smokers, there was no association between arsenic and bladder cancer risk. Conclusions: These, and other data, suggest that ingestion of low to moderate arsenic levels may affect bladder cancer incidence, and that cigarette smoking may act as a co-carcinogen.

Implications of LINE1 Methylation for Bladder Cancer Risk in Women

Purpose: Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood–derived DNA is associated with increased risk of bladder cancer. Experimental Design: LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case-control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation. Results: Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer [95% confidence interval (95% CI), 1.12-2.90] in models controlling for gender, age, and smoking, and the association was stronger in women than in men (odds ratio, 2.48; 95% CI, 1.19-5.17 in women; and odds ratio, 1.47; 95% CI, 0.79-2.74 in men). Among controls, women were more likely to have lower LINE1 methylation than men (P = 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (P = 0.04). Conclusions: LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially among women. Clin Cancer Res; 16(5); 1682–9

Tissue-shrinkage correction factor in the calculation of prostate cancer volume

Many studies that have calculated prostate cancer volumes from microscopic slides have used correction factors, ranging from 1.22 to 1.5, to compensate for tissue shrinkage during tissue processing. We undertook a study to measure tissue shrinkage directly because our experience suggested less shrinkage than that reported by others. Ten prostatectomy specimens were processed in a uniform manner. Multiple identical linear measurements were taken at four stages of processing: in the fresh state, following fixation, following processing, and from the microscopic slide. Linear shrinkage following fixation was minimal (4.1%) but increased to 14.5% following tissue processing. With rehydration and expansion on the flotation bath, tissues swelled so that net linear tissue shrinkage was 4.3%, and net volumetric tissue shrinkage was 12.4%, which translates into a correction factor for tissue shrinkage of 1.14. The following variables had no statistically significant effect on shrinkage: concentration of formalin, whole-mount versus quadrant sections, thickness of tissue slices, length of time in the alcohol dehydration steps, and temperature of the flotation bath over a range of 35 to 45 degrees C. This study suggests that (a) tissue-shrinkage correction factors that have been used in some previous studies may not be applicable for all laboratories because of interlaboratory variations in tissue-processing procedures or differences in measuring shrinkage; and (b) some calculated tumor volumes that have been used for prognostic thresholds may be high because of inflated tissue-shrinkage correction factors.

Epigenetic Inactivation of SFRP Genes and TP53 Alteration Act Jointly as Markers of Invasive Bladder Cancer

In the United States each year, almost 13,000 deaths are attributable to bladder cancer, with the majority of these deaths related to higher stage, muscle-invasive solid tumors. Epigenetic silencing of the secreted frizzled receptor proteins (SFRP), antagonists of the WNT pathway, leads to constitutive WNT signaling, altering cell morphology and motility. Identifying alterations in this pathway in bladder cancer may prove useful for defining the invasive phenotype and provide targets for guiding therapy. Using a population-based study of bladder cancer (n = 355), we examined epigenetic alterations, specifically gene promoter hypermethylation, of four SFRP genes in addition to immunohistochemical staining of TP53, which has been previously shown to be a predictor of invasive disease. We observed a significant linear trend (P < 0.0004) in the magnitude of the risk of invasive disease with the number of SFRP genes methylated. Both TP53 alteration and SFRP gene methylation showed significant independent associations with invasive bladder cancer. Strikingly, in examining the joint effect of these alterations, we observed a >30-fold risk of invasive disease for patients with both altered SFRP gene methylation and intense TP53 staining (odds ratio, 32.1; P < 10(-13)). Overall patient survival was significantly poorer in patients with any SFRP genes methylated (P < 0.0003) and in proportional hazards modeling, patients with methylation of any SFRP gene had significantly poorer overall survival (hazard ratio, 1.78; P < 0.02) controlled for TP53 staining intensity and other survival-associated factors. Classifying tumors based on SFRP methylation status and TP53 protein staining intensity may be a clinically powerful predictor of invasive, deadly disease.

A Case–Control Study of Smoking and Bladder Cancer Risk: Emergent Patterns Over Time

BACKGROUND Cigarette smoking is a well-established risk factor for bladder cancer. The effects of smoking duration, intensity (cigarettes per day), and total exposure (pack-years); smoking cessation; exposure to environmental tobacco smoke; and changes in the composition of tobacco and cigarette design over time on risk of bladder cancer are unclear. METHODS We examined bladder cancer risk in relation to smoking practices based on interview data from a large, population-based case-control study conducted in Maine, New Hampshire, and Vermont from 2001 to 2004 (N = 1170 urothelial carcinoma case patients and 1413 control subjects). We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. To examine changes in smoking-induced bladder cancer risk over time, we compared odds ratios from New Hampshire residents in this study (305 case patients and 335 control subjects) with those from two case-control studies conducted in New Hampshire in 1994-1998 and in 1998-2001 (843 case patients and 1183 control subjects). RESULTS Regular and current cigarette smokers had higher risks of bladder cancer than never-smokers (for regular smokers, OR = 3.0, 95% CI = 2.4 to 3.6; for current smokers, OR = 5.2, 95% CI = 4.0 to 6.6). In New Hampshire, there was a statistically significant increasing trend in smoking-related bladder cancer risk over three consecutive periods (1994-1998, 1998-2001, and 2002-2004) among former smokers (OR = 1.4, 95% CI = 1.0 to 2.0; OR = 2.0, 95% CI = 1.4 to 2.9; and OR = 2.6, 95% CI = 1.7 to 4.0, respectively) and current smokers (OR = 2.9, 95% CI = 2.0 to 4.2; OR = 4.2, 95% CI = 2.8 to 6.3; OR = 5.5, 95% CI = 3.5 to 8.9, respectively) (P for homogeneity of trends over time periods = .04). We also observed that within categories of intensity, odds ratios increased approximately linearly with increasing pack-years smoked, but the slope of the increasing trend declined with increasing intensity. CONCLUSIONS Smoking-related risks of bladder cancer appear to have increased in New Hampshire since the mid-1990s. Based on our modeling of pack-years and intensity, smoking fewer cigarettes over a long time appears more harmful than smoking more cigarettes over a shorter time, for equal total pack-years of cigarettes smoked.

Dendritic Cell Infiltration in Colon Cancer

We quantitatively evaluated dendritic cell (DC) infiltration in primary colorectal cancers from 44 patients and metastatic colorectal tumors from 13 patients using immunohistochemistry for the DC marker CD83, HLA-DR, and the DC activation molecules CD40 and CD86. Nearly all CD83+ cells were also HLA-DR+, CD40+, and CD86+, indicating that the DCs that infiltrate colon cancer in vivo express the activation and costimulatory molecules associated with a mature DC phenotype. The density of DCs in colorectal cancer primaries was three times lower than that seen in normal colonic mucosa (0.29 versus 0.84 CD83+ cells/ high-power field (hpf), p < 0.001). Dendritic cells were rarely observed in metastatic tumors: DC density in metastases was sixfold lower than in colorectal primary tumors (0.05 versus 0.29 CD83+ cells/hpf, p < 0.001). Because cytokines have been shown, in vitro, to exert potent effects on DCs, we also evaluated the relationship between intratumor DC density and the expression of cytokines by tumor-infiltrating lymphocytes (TILs) and tumor cells. Expression of interleukin-10 and transforming growth factor &bgr; by either TIL or tumor cells was not associated with decreased DC density or decreased expression of CD40 or CD86 on DCs. Tumor expression of vascular endothelial growth factor was associated with a more than twofold increase in DC density (p = 0.01). Patients who had a high proportion of TILs expressing tumor necrosis factor (TNF) had a greater intratumor mature DC density than patients with a low proportion of TNF + TIL (0.54 versus 0.21 CD83+ cells/hpf, p < 0.01).

Clinical and immunologic effects of intranodal autologous tumor lysate-dendritic cell vaccine with aldesleukin (interleukin 2) and IFN-α2a therapy in metastatic renal cell carcinoma patients

Purpose: To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-α 2a in metastatic renal cell carcinoma patients. Experimental Design: Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 × 107 cells/1 mL Lactated Ringers solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-α 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti–renal cell carcinoma antibody and cytokine levels. Results: All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4+ T regulatory cells had a strong association with outcome. Pre–IP-10 serum levels approached significance for predicting outcome. Conclusions: The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.

A genome-wide association study of bladder cancer identifies a new susceptibility locus within SLC14A1, a urea transporter gene on chromosome 18q12.3

Genome-wide and candidate-gene association studies of bladder cancer have identified 10 susceptibility loci thus far. We conducted a meta-analysis of two previously published genome-wide scans (4501 cases and 6076 controls of European background) and followed up the most significant association signals [17 single nucleotide polymorphisms (SNPs) in 10 genomic regions] in 1382 cases and 2201 controls from four studies. A combined analysis adjusted for study center, age, sex, and smoking status identified a novel susceptibility locus that mapped to a region of 18q12.3, marked by rs7238033 (P = 8.7 × 10(-9); allelic odds ratio 1.20 with 95% CI: 1.13-1.28) and two highly correlated SNPs, rs10775480/rs10853535 (r(2)= 1.00; P = 8.9 × 10(-9); allelic odds ratio 1.16 with 95% CI: 1.10-1.22). The signal localizes to the solute carrier family 14 member 1 gene, SLC14A1, a urea transporter that regulates cellular osmotic pressure. In the kidney, SLC14A1 regulates urine volume and concentration whereas in erythrocytes it determines the Kidd blood groups. Our findings suggest that genetic variation in SLC14A1 could provide new etiological insights into bladder carcinogenesis.

Chronic lymphocytic leukemia with coexistent Hodgkin's disease. Implications for the origin of the Reed-Sternberg cell.

The association of chronic lymphocytic leukemia (CLL) and Hodgkins disease has been controversial. Pleomorphic lymphoreticular cells resembling Reed-Sternberg cells have been observed in Richters syndrome. Although most observers have favored the view that these cells are a component of a pleomorphic non-Hodgkins lymphoma, some cases of histologically typical Hodgkins disease have been described. We have studied two cases that appear to represent composite CLL and Hodgkins disease, providing evidence for an interrelationship of these two disorders. Classic Reed-Sternberg cells and variants (RS-H) were seen in a backgournd that was otherwise typical of CLL. Both patients initially presented with characteristic findings of CLL in the peripheral blood and bone marrow. The first patient was found to have RS-H cells within lymph nodes at initial presentation, and ultimately progressed to develop a disseminated lymphoma characteristic of Hodgkins disease. In the second patient, RS-H cells were not discovered until 5 years later. Immunophenotypic studies confirmed these morphologic impressions. The predominant lymphocyte population had a phenotype consistent with B-cell CLL. By contrast, the RS-H cells were strongly positive for CD15 (Leu M1) with staining of the Golgi region and cell membrane. Additionally, the RS-H cells were surrounded by rosettes of lymphocytes that marked as T cells. In both of the patients, a small percentage of RS-H cells expressed postivity for the B-cell marker L-26, which may indicate an origin from the underlying CLL. These findings support a B-cell origin for the malignant cell in some cases of Hodgkins disease adn suggest taht Hodgkins disease in some patients may be related to or derived from a coexisting lymphoid malignancy.