Claudia Veggiani

Kidney and Urinary Tract Polyomavirus Infection and Distribution Molecular Biology Investigation of 10 Consecutive Autopsies

CONTEXT Distinct human polyomavirus genotypes cause different diseases in patients with renal transplants: BK virus (BKV) causes tubulointerstitial nephritis and ureteral stenosis, whereas both JC virus (JCV) and BKV are responsible for hemorrhagic cystitis. These findings could result from a selective infection of kidney and urinary tract segments by JCV or BKV. OBJECTIVE To verify this hypothesis, 10 complete, unselected, consecutive autopsies from 9 immunocompetent patients and 1 patient affected by acquired immunodeficiency syndrome were investigated. DESIGN Samples from kidneys (n = 80), renal pelvis (n = 20), ureter (n = 40), and urinary bladder (n = 30) obtained from 10 consecutive autopsies were investigated by means of multiplex nested polymerase chain reaction to detect polyomavirus DNA and to distinguish different species of the Polyomavirus genus. In situ hybridization and immunohistochemistry were also carried out to define the viral status of the infected tissues. RESULTS Polyomavirus DNA was detected in all of the subjects (positive samples ranging from 2 to 7 samples), for a total of 43 of 170 samples (25.3%), distributed as follows: urinary bladder (10/30, 33%), renal pelvis (6/20, 30%), ureter (10/40, 25%), and kidney tissue (17/80, 21%). We found that JCV was most frequently detected overall (23/43 samples, 53.5%) and was also detected most frequently within the kidney (8/17 positive samples, 47%), the renal pelvis (5/6 positive samples, 70%), and the ureter (7/10 positive samples, 70%), whereas BKV was found in 14 samples (32.5%), and it was the prevailing genotype in urinary bladder (6/10 positive samples, 60%). Coinfection of BKV-JCV was found in 6 samples (14%). Immunohistochemistry and in situ hybridization returned negative results. CONCLUSIONS The viruses JCV and BKV latently persist randomly in kidney and urinary tract. Distinct diseases induced by them could be related more closely to molecular viral rearrangements than to the topographic distribution of latent viruses.

Presence and expression of JCV early gene large T Antigen in the brains of immunocompromised and immunocompetent individuals

JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV‐positive PML patients, 15 HIV‐positive patients with other neurological diseases, 15 HIV‐positive patients without neurological disorders, and 15 HIV‐negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV‐positive brains (with and without neurological diseases), and 1 of 15 HIV‐negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV‐negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML. J. Med. Virol. 80:2147–2152, 2008.

Periodic Assessment of Urine and Serum by Cytology and Molecular Biology as a Diagnostic Tool for BK Virus Nephropathy in Renal Transplant Patients

OBJECTIVE To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.

Epidermal Growth Factor Receptor Gene Analysis With a Highly Sensitive Molecular Assay in Routine Cytologic Specimens of Lung Adenocarcinoma

Epidermal growth factor receptor (EGFR) gene mutational analysis is critical for guiding the treatment of lung adenocarcinoma. In everyday clinical practice, EGFR testing is frequently centralized in referral laboratories that may receive paucicellular cytologic specimens, often fixed in various ways. We conducted a search for EGFR mutations in 108 cytologic samples of lung adenocarcinoma from different hospitals using the TheraScreen EGFR29 kit. These samples included 80 (74.1%) fine-needle aspirations, 13 (12%) pleural/ascitic fluids, 13 (12%) bronchial washings, and 2 bronchial brushings. The samples were fixed in ethanol (n = 79), Duboscq-Brasil (n = 18) or formalin (n = 10); 1 was unfixed. Ninety-two (85.2%) were amplified, 16 (14.8%) were not. Mutations were detected in 22 (23.9%) of 92 amplified samples, 9 containing less than 200 cancer cells, and 4 with less than 50% cancer cells. DNA was amplified in 12 of 18 Duboscq-Brasil-fixed samples. These findings indicate that cytologic specimens are adequate for EGFR testing when a highly sensitive assay is used, even if they are paucicellular or not optimally fixed.

Latent human polyomavirus infection in pregnancy: investigation of possible transplacental transmission

Aims: The purpose of the study was to investigate the transplacental transmission of the human polyomaviruses JCV and BKV. Methods: Urine and blood samples from 300 pregnant women underwent cytological analysis to search for ‘decoy cells’, nested PCR to identify presence and genotype of isolated polyomaviruses, and sequence analysis of the transcription control region. Nested PCR was also used to study the umbilical cord blood of all their newborns. Results: Decoy cells were identified in only one urine sample (1/300; 0.33%); polyomavirus DNA was detected in 80 urine samples (26.6%) corresponding to BKV alone in 28 samples (9.3%), JCV alone in 49 samples (16.3%) and both JCV‐BKV in three samples (1%). Blood samples were positive in 17 cases (5.6%), corresponding to BKV alone in 10 (3.3%), and JCV alone in 7 (2.3%). Rearrangements of the transcription control region were found in only one urinary JCV strain, consisting of the insertion of 13 bp at D block, whereas point mutations were identified in 11 BKV and 11 JCV strains detected from urine. Sequence analysis of the BKV strains detected in blood samples revealed a 20 bp insertion of P block (P42–61) in human chromosomes 20 (five cases) and 14 (three cases); two JCV strains had single bp point mutations. The search for polyomavirus DNA in umbilical cord blood samples was always negative. Conclusions: Polyomavirus DNA was frequently detected in pregnancy, whereas genomic rearrangements were rare, and no evidence of transplacental transmission of polyomavirus was obtained.

Genomic mutations of viral protein 1 and BK virus nephropathy in kidney transplant recipients

Genomic variability in the viral protein 1 region of BK polyomavirus (BKV) may change the ability of the virus to replicate. The significance of such changes was studied in clinical samples taken from kidney transplant patients with and without BKV nephropathy. A 94 base‐pair fragment of viral protein 1 was amplified from 68 urine, 28 blood, and 12 renal biopsy samples from eight patients with BKV nephropathy, and from 100 urine samples, 17 blood and three renal biopsy samples from 41 of 218 controls. The DNA was sequenced and the amino acid changes were predicted by the Expert Protein Analysis System program (ExPASy, Swiss Institute of Bioinformatics, Geneva, Switzerland). Single base‐pair mutations were detected more frequently in the samples from the BKV nephropathy patients than in the controls, and this was the only statistically significant finding of the study (P < 0.05), thus suggesting a greater genetic instability in BKV nephropathy associated strains. The amino acid changes were distributed at random in both BKV nephropathy patients and controls. However, one aspartic acid‐to‐asparagine substitution at residue 75 was detected in all samples of the one patient with BKV‐associated nephropathy, who developed disease progression confirmed by histology, and not in any of the other patient or control samples. Whether this specific amino acid change plays a role in disease deserves further study. J. Med. Virol. 81:1385–1393, 2009.

Fluorescence in situ hybridisation in the cytological diagnosis of pancreatobiliary tumours

Aims: To assess the sensitivity, specificity, positive and negative predictive values of fluorescence in situ hybridisation (FISH) and conventional cytology in identifying bile duct stricture malignancies. Methods: Brushing samples were collected from 64 patients by means of endoscopic retrograde cholangiopancreatography, and assessed cytologically and by means of a multi-probe FISH set. The cytological diagnoses were: positive, negative and suspicious, whereas criteria for FISH positivity were: more than five polysomic cells or more than 10 trisomic cells for chromosomes 3 or 7. Results: Forty-eight of the 64 patients showed histological or clinical signs of malignancy. The sensitivity of cytology was high (77%) if suspicious cases were considered positive, but was significantly lower than that of FISH if suspicious cases were considered negative (58% versus 90%; p < 0.05). The specificity of cytology was 81% (positive and suspicious) or 100% (negative and suspicious), and the specificity of FISH was 94% (p = 1). FISH yielded one false negative result (isolated chromosome 7 trisomy). FISH allowed a definite diagnosis of 9/12 cytologically inconclusive cases. Conclusions: Our findings suggest using FISH in the case of bile duct strictures cytologically negative or inconclusive; a FISH diagnosis of malignancy should only be made in the presence of polysomic pattern.

BK virus sequences in specimens from aborted fetuses

Given the conflicting results of the few published studies, the aim of this retrospective molecular‐based study of 10 aborted fetuses that underwent complete autopsy and 10 placentas was carried out to determine whether BK polyomavirus (BKV) can be transmitted transplacentally. The interruption of pregnancy was due to a miscarriage (five cases) or a prenatal diagnosis of severe intrauterine malformations (five cases). Samples from the brain, heart, lung, thymus, liver, and kidney were taken from each fetus, and two samples were obtained from all of the placentas. The presence of BKV was investigated by means of PCR using primers specific for the transcription control region (TCR) and viral capsidic protein 1 (VP1) and DNA extracted from formalin‐fixed, paraffin‐embedded tissue. BKV genome was detected in 22 of 60 samples (36.6%) from seven fetuses (70%), regardless of the cause of abortion: VP1 was amplified in 12 samples (54%), TCR in seven (32%), and both in three (14%). VP1 was also detected in one placental sample. BKV sequences were most frequently detected in heart and lung (five cases), but sequence analyses of TCR and VP1 revealed a high degree of genomic variability among the samples taken from different organs and the placenta. These results indicate that BKV can cross the placenta during pregnancy and become latent in fetal organs other than the kidney and brain (previously considered the main targets of BKV latency). This may happen in early pregnancy and does not seem to be associated with an increased risk of abortion. J. Med. Virol. 82:2127–2132, 2010.

Are Sequence Variations in the BK Virus Control Region Essential for the Development of Polyomavirus Nephropathy

BK virus replication is regulated by the noncoding control region (NCCR); major NCCR rearrangements could modify the strength of viral replication, having a role in the development of polyomavirus-associated nephropathy (PAN). Urine (n = 34), blood (n = 32), and renal biopsy samples (n = 13) from 5 transplant recipients with PAN underwent nested polymerase chain reaction to search for the NCCR region. Sequence analysis was performed on all NCCR fragments obtained. Decoy cells were evaluated semiquantitatively in urine and PAN staged in renal biopsy specimens; the results were related to the presence and type of NCCR sequence variations. Major NCCR rearrangements were found in urine (9/75 [12%]), blood (7/30 [23%]), and renal biopsy (4/15 [27%]) samples in 3 cases; 2 cases had only unrearranged strains. Neither the detection and number of decoy cells nor the PAN stage were related to the specific type of NCCR sequence rearrangements. NCCR rearrangements do not seem essential for the development of PAN.

KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance

Mutations of KRAS are detectable in 70-90% of pancreatic duct adenocarcinomas (PDAC), using direct sequencing. We used a highly sensitive molecular method in order to investigate: (a) the frequency and prognostic significance of different KRAS mutations and, (b) whether the presence of KRAS mutations in histologically-negative resection margins of PDAC could explain local tumor recurrence after surgery. Twenty-seven patients with histologic diagnosis of PDAC, radical pancreaticoduodenectomy and histologically-negative margins were evaluated. KRAS mutations were searched for mutant-enriched PCR in tumor and negative resection margins. KRAS mutations were detected in 85.2% of the cases; the most frequent mutation was G12D (48.1%). Shorter OS was found in patients with G12D (25 months; 95% CI, 20.5-29.5), vs patients with other mutations (31.5 months; 95% CI, 25.6-37.1) (N.S.). KRAS mutation in histologically-negative margins was detected in one patient who died of locoregional recurrence; six patients had tumor recurrence but no mutations in surgical margins. The high frequency of KRAS mutations suggests a search for KRAS status to improve the diagnosis in suspected cases; the G12D mutation could be related to poor prognosis, but without statistical significance. No correlation was found between the frequency of cancer recurrence and KRAS mutations in surgical margins.