Sydney D. Finkelstein

Gene Expression Alterations in Prostate Cancer Predicting Tumor Aggression and Preceding Development of Malignancy

PURPOSE The incidence of prostate cancer is frequent, occurring in almost one-third of men older than 45 years. Only a fraction of the cases reach the stages displaying clinical significance. Despite the advances in our understanding of prostate carcinogenesis and disease progression, our knowledge of this disease is still fragmented. Identification of the genes and patterns of gene expression will provide a more cohesive picture of prostate cancer biology. PATIENTS AND METHODS In this study, we performed a comprehensive gene expression analysis on 152 human samples including prostate cancer tissues, prostate tissues adjacent to tumor, and organ donor prostate tissues, obtained from men of various ages, using the Affymetrix (Santa Clara, CA) U95a, U95b, and U95c chip sets (37,777 genes and expression sequence tags). RESULTS Our results confirm an alteration of gene expression in prostate cancer when comparing with nontumor adjacent prostate tissues. However, our study also indicates that the gene expression pattern in tissues adjacent to cancer is so substantially altered that it resembles a cancer field effect. CONCLUSION We also found that gene expression patterns can be used to predict the aggressiveness of prostate cancer using a novel model.

Human polyoma virus-associated interstitial nephritis in the allograft kidney

BACKGROUND Asymptomatic polyoma virus infection documented by urine cytology or serology is well known, but the clinical course of biopsy-proven interstitial nephritis is not well defined. METHODS Twenty-two cases were identified by histology, immunostaining, in situ hybridization, electron microscopy, or polymerase chain reaction. RESULTS The clinical features mimicked acute rejection (n=19), chronic rejection with incidental diagnosis at nephrectomy (n=2), or drug toxicity (n=1). Histology showed homogenous intranuclear inclusions. In situ hybridization showed BK virus (BKV) to be the predominant species, but polymerase chain reaction documented JC virus co-infection in one of five cases so tested. Electron microscopy in seven cases showed 20-51-nm virions. The two cases diagnosed at nephrectomy received no therapy. Initial antirejection therapy in 12 cases led to clearance of the virus in 1/12 (8%), partial therapeutic response in 3/12 (25%), and graft loss in 8/12 (67%) cases. The last recorded creatinine in patients with functional grafts ranged from 1.9 to 7.0 (median: 4.5) mg/dl, 0.4-45 (median: 4.0) months after initial diagnosis. The remaining eight cases treated by reduction of immunosuppression at the outset have been free of graft loss for 0.2-10.0 (median: 4.8) months since diagnosis, and clearance of virus has been documented in three of six (50%) cases. The serum creatinine in these patients is 1.7-6.0 (median: 2.4) mg/dl, 0.2-10 (median: 4.8) months after diagnosis. Follow-up biopsies performed 1-23.5 months after diagnosis show chronic allograft nephropathy. CONCLUSIONS Polyoma virus tubulo-interstitial nephritis-associated graft dysfunction usually calls for judicious decrease in immunosuppression and monitoring for acute rejection. Development of methods to serially quantify the viral load in individual patients could potentially improve clinical outcome.

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study

BACKGROUND The role of pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts remains unclear. OBJECTIVE Our purpose was to evaluate the utility of a detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts. DESIGN Prospective, multicenter study. PATIENTS Patients with pancreatic cysts presenting for EUS evaluation. INTERVENTION EUS-guided pancreatic cyst aspirates cytology evaluation, carcinoembryonic antigen (CEA) level determination, and a detailed DNA analysis; incorporating DNA quantification, k-ras mutation and multiple allelic loss analysis, mutational amplitude, and sequence determination. MAIN OUTCOME MEASUREMENTS Cyst fluid analysis compared with surgical pathologic or malignant cytologic examination. RESULTS The study cohort consisted of 113 patients with 40 malignant, 48 premalignant, and 25 benign cysts. Cyst fluid k-ras mutation was helpful in the diagnosis of mucinous cysts (odds ratio 20.9, specificity 96%), whereas receiver-operator characteristic curve analysis indicated optimal cutoff points for allelic loss amplitude (area under the curve [AUC] 0.79; optimal value > 65%) and CEA (AUC 0.74; optimal value >148 ng/mL). Components of DNA analysis detecting malignant cysts included allelic loss amplitude over 82% (AUC 0.9) and high DNA amount (optical density ratio >10, AUC 0.79). The criteria of a high amplitude k-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. All malignant cysts with negative cytologic evaluation (10/40) could be diagnosed as malignant by using DNA analysis. LIMITATIONS Limited follow-up, selection bias. CONCLUSIONS Elevated amounts of pancreatic cyst fluid DNA, high-amplitude mutations, and specific mutation acquisition sequences are indicators of malignancy. The presence of a k-ras mutation is also indicative of a mucinous cyst. DNA analysis should be considered when cyst cytologic examination is negative for malignancy.

Gene expression analysis of prostate cancers.

Prostate cancer is a biologically heterogeneous disease with considerable variation in clinical aggressiveness. The behavior of prostate cancer can be considered a direct or indirect result of aberrant alterations of gene expression in prostate epithelial cells. Identification of the patterns of gene‐expression alterations that are related to the aggressiveness of prostate cancers will greatly assist the development of tools for early detection of prostate cancers with poor clinical outcome and identification of targets for future therapeutic intervention. To detect the patterns of gene‐expression alterations of prostate cancers, we performed a comprehensive gene‐expression analysis on 30 prostate tissues of various levels of invasiveness (ranging from those confined to the organ to distant metastases) and Gleason grades (combined scores 4–9), using the Affymetrix chip set Hu35k (A–D) and U95a. Following three sequential selection screens, we identified 84 largely novel genes and expressed sequence tag (EST) sequences whose expression levels were altered significantly in prostate cancer samples compared with control normal tissues. In addition, the expression levels of a group of 12 genes and EST sequences was found to be altered significantly in aggressive type of prostate cancers but not in organ‐confined prostate cancers. Cluster analysis using the 84‐gene list showed that the highly aggressive prostate cancers contained gene‐expression patterns that were distinct from organ‐confined prostate cancers.

Correlates of Quantitative Measurement of BK Polyomavirus (BKV) DNA with Clinical Course of BKV Infection in Renal Transplant Patients

ABSTRACT BK virus-allograft nephropathy (BKVAN) is an increasingly recognized complication after kidney transplantation. Quantitative tests have been advocated to monitor patients, but data demonstrating their efficacy are relatively limited. We developed a real-time PCR assay to quantitate BK virus loads in the setting of renal transplantation, and we correlated the BK virus load with clinical course and with the presence of BK virus in renal biopsy specimens. BK virus loads were measured in urine, plasma, and kidney biopsy samples in three clinical settings: (i) patients with asymptomatic BK viruria, (ii) patients with active BKVAN, and (iii) patients with resolved BKVAN. Active BKVAN was associated with BK viremia greater than 5 × 103 copies/ml and with BK viruria greater than 107 copies/ml in all cases. Resolution of nephropathy led to resolution of viremia, decreased viruria levels, and disappearance of viral inclusions, but low-level viral DNA persisted in biopsy specimens even for patients whose viruria was cleared. All but one patient in the resolved BKVAN group carried a urinary viral load below 107 copies/ml. Viral loads in patients with asymptomatic viruria were generally lower but in some cases overlapped with levels more typical of BKVAN. One patient with asymptomatic viruria and with a viral load overlapping values seen in BKVAN had developed nephropathy by the time of follow-up. In conclusion, serial measurement of viral loads by quantitative PCR is a useful tool in monitoring the course of BK virus infection. The results should be interpreted in conjunction with the clinical picture and biopsy findings.

The Role of Pancreatic Cyst Fluid Molecular Analysis in Predicting Cyst Pathology

BACKGROUND & AIMS Current methods to detect malignancy in mucinous cystic neoplasms of the pancreas remain inadequate. The role of detailed molecular analysis in this context was investigated. METHODS Endoscopic ultrasound-guided pancreatic cyst aspirates were prospectively collected during a period of 19 months and studied for cytology, carcinoembryonic antigen level, and molecular analysis. Molecular evaluation incorporated DNA quantification (amount and quality), k-ras point mutation, and broad panel tumor suppressor linked microsatellite marker allelic loss analysis by using fluorescent capillary electrophoresis. The sequence of mutation acquisition was also calculated on the basis of a clonal expansion model, and comparison was made to the final pathology. RESULTS Thirty-six cysts with confirmed histology were analyzed. There were 11 malignant, 15 premalignant, and 10 benign cysts. Malignant cysts could be differentiated from premalignant cysts on the basis of fluid carcinoembryonic antigen level (P=.034), DNA quality (P=.009), number of mutations (P=.002), and on the sequence of mutations acquired (P<.001). Early k-ras mutation followed by allelic loss was the most predictive of a malignant cyst (sensitivity, 91%; specificity, 93%). CONCLUSIONS Malignant cyst fluid contains adequate DNA to allow mutational analysis. A first hit k-ras mutation followed by allelic loss is most predictive of the presence of malignancy in a pancreatic cyst. This approach should serve as an ancillary tool to the conventional work-up of pancreatic cysts. Cumulative amount and timing of detectable mutational damage can assist in diagnosis and clinical management.

Carcinosarcomas (malignant mixed mullerian tumors) of the female genital tract: comparative molecular analysis of epithelial and mesenchymal components.

Female genital tract carcinosarcomas (FGTCS) are biphasic neoplasms composed of an admixture of malignant epithelial and mesenchymal elements. Histogenesis of FGTCS centers on two theories: (1) simultaneous formation of independent tumors (biclonal theory), (2) multidirectional differentiation of a single neoplasm (monoclonal theory). In an attempt to resolve this histogenetic controversy, we determined the presence, specific genotype, and timing of p53 mutational change in each component of FGTCS using a topographic genotyping (TG) approach. We selected 43 FGTCS from the files of Magee-Womens Hospital, Pittsburgh, and initially immunostained them for p53 protein. Strong p53 immunopositivity was detected in 35 (82%) of 43 tumors. Subsequently, topographic genotyping (TG) was performed on a subset of nine immunopositive tumors with sufficiently distinct malignant components to enable effective sampling. All nine tumors showed point mutations in p53 exons 5 through 8. In each case, the identical point mutational genotype was present in both components. Furthermore, in all nine cases mutations were present with loss of the wild-type allele. P53 gene mutation is a frequent event in progression of FGTCS. Of importance, both p53 mutation and allelic loss occur before the differentiation into separate epithelial and mesenchymal malignant components. These molecular findings strongly support monoclonal, multidirectional histogenesis of FGTCS.

Molecular analysis to demonstrate that odontogenic keratocysts are neoplastic.

CONTEXT Odontogenic keratocysts (OKCs) are unique odontogenic lesions that have the potential to behave aggressively, that can recur, and that can be associated with the nevoid basal cell carcinoma syndrome. Whether they are developmental or neoplastic continues to be debated. OBJECTIVES To identify loss of heterozygosity of tumor suppressor genes in OKCs and to suggest a pathogenetic origin for these lesions. DESIGN We examined 10 OKCs for loss of heterozygosity of tumor suppressor genes, using a microdissection and semiquantitative genotyping analysis. The genes analyzed included 10 common tumor suppressor genes, as well as the PTCH gene, which is mutated in nevoid basal cell carcinoma syndrome. RESULTS Loss of heterozygosity was seen in 7 of 10 cases, with a frequency between 11% and 80% of the genes studied. The genes that exhibited the most frequent allelic losses were p16, p53, PTCH, and MCC (75%, 66%, 60%, and 60%, respectively). Daughter cysts were associated with a higher frequency of allelic loss (P =.02), but epithelial budding was not. CONCLUSIONS Our study indicates that a significant number of OKCs show clonal loss of heterozygosity of common tumor suppressor genes. The finding of clonal deletion mutations of genomic DNA in these cysts supports the hypothesis that they are neoplastic rather than developmental in origin.

Generation of T Cells Specific for the Wild-Type Sequence p53 264–272 Peptide in Cancer Patients: Implications for Immunoselection of Epitope Loss Variants

Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53264–272 epitope to generate CTL from circulating precursor T cells of HLA-A2.1+ healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53264–272 peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53264–272 peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53264–272 epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53264–272 epitope. These findings suggest that in vivo, CTL specific for the wt p53264–272 peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.

Quantitation of viral DNA in renal allograft tissue from patients with BK virus nephropathy.

Background. BK virus (BKV) allograft nephropathy (BKVAN) is a complication in renal transplantation recipients. Histopathology is the gold standard for diagnosis. Quantitative polymerase chain reaction (PCR) assay for renal biopsy has not been evaluated as a diagnostic test. Determination of renal BKV load may identify patients at risk for disease before histologic nephropathy. Methods. Quantitative PCR assay for BKV DNA was performed in 28 biopsies of patients with BKVAN; 50 biopsies were performed before a diagnosis of BKVAN, and 126 control biopsies were from patients without a history of BKVAN. Results. BKV DNA was present in 19 of 50 (38%) biopsies performed 1 to 164 weeks before diagnosis of BKVAN. The viral load (mean 216 copies/cell) was lower than in biopsies of patients with BKVAN (mean 6063 viral copies/cell, P <0.05). In 10 of 127 (7.8%) control biopsies, a low level of BKV DNA (mean 3.8 copies/cell) was found in three biopsies from chronic allograft nephropathy patients; two biopsies with acute rejection; four biopsies with borderline change; and one biopsy with cytomegalovirus nephritis. Conclusion. BKV load exceeding 59 copies per cell identified all cases of BKVAN. The diagnostic sensitivity, specificity, positive predictive value, and negative predictive value of quantitative PCR were 100%, 92.1%, 73.6%, and 100%, respectively. Lower levels of BKV DNA were identified in biopsies performed before viral nephropathy development. Future research will determine if earlier recognition of at-risk patients allows application of antiviral strategies to improve graft outcome.