Patricia A. Swalsky

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.

p53 sequence analysis predicts treatment response and outcome of patients with esophageal carcinoma

The ability to predict biologic behavior and treatment responsiveness would be a valuable asset in the multimodality approach to esophageal carcinoma. The authors examined whether alterations of the p53 gene correlate with clinicopathologic parameters, response to preoperative chemotherapy/radiotherapy, and outcome in patients with esophageal carcinoma.

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.

Molecular pathogenesis of pulmonary carcinosarcoma as determined by microdissection-based allelotyping.

Pulmonary carcinosarcoma is a rare, biphasic tumor composed of malignant epithelial and mesenchymal elements. Its histogenesis is controversial in light of the presence of divergent cell lineages and the clonal nature of malignancy. To address these issues, we performed an extensive comparative genotypic analysis using microdissection to secure representative mesenchymal and epithelial components from each of six cases of pulmonary carcinosarcoma. Loss of heterozygosity was analyzed with a panel of 12 polymorphic microsatellite markers designed to indicate allelic loss and situated in proximity to known tumor suppressor genes located on 1p, 3p, 5q, 9p, 10q, and 17p. In accordance with the relatively greater biologic aggressiveness of this tumor type, both the epithelial and mesenchymal components showed extensive allelic loss, most notably for 3p, 5q, and 17p. More importantly, we found overall equivalent patterns of acquired allelic loss between the two components on an individual case basis, strongly supporting the monoclonal origin of these neoplasms. Minor differences in the allelic fingerprint between the two cell lineages could be explained by progressive accumulation of allelic loss alterations that appear to occur more frequently in the mesenchymal component of the tumor. The data support the efficacy of microdissection-based allelic fingerprinting to delineate the relationship between different morphologic components of a single neoplasm.

Prognostic value of TP53 and K-ras-2 mutational analysis in stage III carcinoma of the colon

Background Genetic mutations involving onccgenes and tumor-suppressor genes occur in carcinogenesis, and may affect biologic behavior of neoplasms. In this study, we analyzed the prognostic value of mutational analysis in Colon carcinoma. Patients and methods Archival pathology specimens from 70 consecutive patients, resected for stage III colon carcinoma, were analyzed for point mutations by amplification and direct sequencing of exons from the K- ras -2 and the TP53 genes (topographic genotyping). Mutations were compared with adverse histopathologic features (poor differentiation, vascular and lymphatic invasion, mucin production) as prognostic markers. Results Five-year survival was 75% in patients with nonmutated lesions, significantly lower (21%) with TP53 mutations ( P = 0.01), and intermediate with K- ras -2 only (45%) or both K- ras -2 and TP53 mutations (36%). A TP53 mutation carried the highest relative risk of death (2.39; 95% confidence interval, 1.29 to 4.42; P = 0.006). There was an additive effect on the risk of death between TP53 mutations and adverse histopathologic features. Conclusions The information derived from mutational analysis is creating new prognostic variables that may play a role in the choice of therapy for colorectal carcinoma.

Use of microsatellite marker loss of heterozygosity in accurate diagnosis of pancreaticobiliary malignancy from brush cytology samples

Background: Brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. Aim: To improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumour suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. Methods: Twenty six patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to six tumour suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and microdissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR). PCR products were compared for LOH and k-ras codon 12 mutations. Results: Seventeen patients were confirmed to have pancreaticobiliary adenocarcinoma. Nine patients had benign strictures (eight proven surgically, one by follow up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and microdissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from nine cases without cancer carried no LOH (p<0.001). LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p<0.001). Conclusion: LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminates reactive from malignant cells, with 100% sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumour likely reflect intratumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.

DNA sequencing of viral capsid protein VP-1 region in patients with BK virus interstitial nephritis

Background. Mutations in the viral capsid protein VP-1 region are associated with increased pathogenicity of polyomavirus in experimental systems. This study sought to determine whether analogous viral genetic changes occur in human BK virus (BKV) interstitial nephritis (ISN). Methods. PCR was used to amplify a 94-bp nucleotide sequence of the viral capsid protein VP-1 region (positions 1740–1833, Dun numbering) in 49 biopsies obtained from 24 patients with BKV-ISN. DNA sequencing was performed by the dideoxy method. Result. The VP-1 region was highly polymorphic and 22 “hot spots” of sequence variability were noted. Genotypes I, II, and IV were assigned to 13, 1, and 5 cases, respectively, but 5 cases could not be unambiguously classified due to sequence heterogeneity at sites used to discriminate between genotypes. Even in cases where genotypes could be assigned, only 5 biopsies showed complete sequence identity with published genotype sequences. Sequential biopsies showed temporal changes in one or more nucleotides in all patients with multiple samples. In one patient, the initial biopsy showed viral genotype 1, although subsequent biopsies showed complex genetic patterns, including a biopsy consistent with viral genotype IV. Conclusions. Many viral strains associated with BKV-ISN are difficult to classify and possibly distinct from those described in kidney transplant recipients without BKV-ISN. VP-1 sequences undergo continual modification as patients are followed in time. This genetic instability could conceivably have implications for evasion of host immunity and development of resistance to antiviral drugs.