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Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level. [ABSTRACT FROM AUTHOR]

Abstract Clinical diagnosis of kidney transplants related illnesses is not a simple task. Several studies were conducted to define diseases and complications after renal transplantation, but there are no comprehensive guidelines about diagnostic tools for their prevention and detection. The Authors of this review looked for the medical literature and pertinent publications in particular to understand the role of Human Polyomavirus BK (BKV) in renal failure and to recognize analytical techniques for BK virus associated nephropathy (BKVAN) detection.

Context.--BK virus strains or regulatory region sequence variations may play a role in the pathogenesis of polyomavirus-associated nephropathy (PVAN), although no definite relationship has yet been demonstrated. Objective.--To investigate the pathologic significance of BK virus strains and regulatory region sequence variations. Design.--Eight (3.5%) of 226 patients with renal transplants developed PVAN; the remaining 218 cases were used as controls. From the patients who developed PVAN, 70 urine samples, 63 blood samples, and 17 renal biopsy samples were taken, and 682 urine samples, 677 blood samples, and 101 renal biopsy samples were taken from the control cases. Amplification and sequence analyses of regulatory region were obtained, and the sequences were analyzed using the Basic Local Alignment Search Tool program. Results.--The WWT strain was more frequently detected in PVAN cases than in the control cases (urine: 88.5% vs 22.1%; blood: 85.2% vs 40%; renal biopsies: 77.8% vs 0%), and the AS and WW strains were only isolated from controls. Strain 128-1 was frequently associated with JC virus coinfection in both groups (PVAN: 78.3%; controls: 98%). Major WWT rearrangements were detected in 29.6% of the urine samples, 30.4% of the blood samples, and one renal biopsy from the PVAN cases, but in only one urine sample from the controls. Insertion of 8 base pairs (P block) was found in all 128-1 strains; WW and AS were archetypal in 78.9% and 57.7% of the samples, respectively. Conclusions.--Although the study included only 8 PVAN cases, regulatory region sequence variations seem to be frequent and independent of the development of the disease, and the WWT strain seems more frequently related to the development of nephropathy than other strains., Polyomavirus-associated nephropathy (PVAN) is a tubular interstitial inflammatory disease caused by lytic infection of epithelial tubular cells by human BK polyomavirus (BKV) in patients who have undergone kidney transplantation. First [...]

Polyomavirus-associated nephropathy (PVAN) is a major complication that occurs after renal transplantation and is induced by reactivation of the human polyomavirus BK (BKV). The structure of the viral capsid protein 1 (VP1) is characterized by the presence of external loops, BC, DE, EF, GH, and HI, which are involved in receptor binding. The pathogenesis of PVAN is not well understood, but viral risk factors are thought to play a crucial role in the onset of this pathology. In an attempt to better understand PVAN pathogenesis, the BKV-VP1 coding region was amplified, cloned, and sequenced from the urine of kidney transplant recipients who did, and did not, develop the pathology. Urine viral loads were determined by using real time quantitative PCR (Q-PCR). Amino acid substitutions were detected in 6/8 patients, and 6/7 controls. The BC and EF loop regions were most frequently affected by mutations, while no mutations were found within the GH and HI loops of both patients and controls. Some mutations, that were exclusively detected in the urine of PVAN patients, overlapped with previously reported mutations, although a correlation between changes in amino acids and the development of PVAN was not found. Urine viral loads were higher than that of the proposed cut-off loads for identification of patients that are at a high risk of developing PVAN (107 copies/ml), both in the PVAN and control groups, thus confirming that urine viral load is not a useful predictive marker for the development of PVAN. J. Cell. Physiol. 222:195–199, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Mutation analysis of KRAS is needed before starting treatment with anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer (CRC). In most of the cases, testing is performed on primary tumors, assuming that KRAS mutation status does not change in metastasis although correlation studies gave conflicting results. We evaluated the KRAS status concordance rate between primary tumors and related metastasis using a highly sensitive molecular assay. Forty-five primary tumors and related metastases from patients with CRC (28/45 male-62.2% and 17/45 female-37.8%; mean age 66.4 years) were analyzed by using TheraScreen: KRAS mutational kit. Metastatic samples were collected from lymph nodes (8/45-17.8%) and visceral sites (37/45-82.2%); 23 were synchronous (49%) and 22 were metachronous (51%), obtained after a mean of 30.8 months after the first diagnosis of CRC. Twenty-eight patients had KRAS mutations in both primary CRC and related metastases (62.2%). No differences in type and frequency of mutations were identified, despite different metastatic sites and time of onset of metastatic disease. Our results indicate that the mutation status of KRAS is the same in primary CRC and metastasis, suggesting that in clinical practice, KRAS testing can be performed on both tumor tissues when using a highly sensitive molecular assay., (Copyright © 2013 Elsevier GmbH. All rights reserved.)