Analysis of major BCR-ABL1 mRNA by digital polymerase chain reaction is useful for prediction of international scale.

BACKGROUND: Major BCR-ABL1 mRNA in patients with chronic myeloid leukemia (CML) has generally been analysed by real-time polymerase chain reaction (PCR). Application of the international scale (IS) for the quantification of major BCR-ABL1 mRNA has been recommended in several sets of guidelines, including those of the European LeukemiaNet. The aim of this study was to clarify the efficacy of digital PCR technology for the IS of BCR-ABL1 mRNA in the patients with CML by comparing with real-time PCR. METHODS: The analysis of BCR-ABL1 mRNA was carried out by the Ipsogen® BCR-ABL1 Mbcr IS-MMR DX Kit (Qiagen), and the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific ) using 20 peripheral blood samples obtained from the 9 patients with CML at Sapporo Medical University Hospital. RESULTS: The correlation between the data obtained by digital PCR and by real-time PCR was really high at R = 0.96. The detection limit of digital PCR was up to 0.003% and was equal to IS with 0.01% or less in comparison with real-time PCR. CONCLUSIONS: Digital PCR technology is promising for predicting the IS value with similar efficacy to real-time PCR and should be useful for simple monitoring of the effects of tyrosine kinase inhibitor (TKI) treatments.

Measurable impact of RNA quality on gene expression results from quantitative PCR.

Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise of microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5'-3' difference in quantification cycle (Cq) and HPRT1 3' Cq value based on a 5'/3' ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences and a normalization factor based on the mean expression level of four reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of reverse transcription quantitative PCR based results in relation to RNA quality.

Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

BACKGROUND: Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). METHODS: DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. RESULTS: Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p < .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p < .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p < .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. CONCLUSION: DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Correcting false gene expression measurements from degraded RNA using RTQ-PCR.

This paper describes a method allowing correcting false gene expression measured on highly degraded RNA using real-time quantitative reverse transcription-polymerase chain reaction (RTQ-PCR). RNA was isolated from different models (in vitro cell lines, in vivo models of human and dog) and different tissue types. In vitro RNA degradation and modeling of in vivo degradation were applied on intact and degraded total RNA. Gene expression (eg, Bcl-2, GAPDH, PGK, PSME3, RAB2, BAX) was measured using RTQ-PCR. 18S rRNA proved to be the most constant house-keeping gene. Less than 10-fold degraded RNA can be quantified correctly when using 18S rRNA for normalization purposes. Higher-fold degraded RNA can be quantified correctly up to a precision that is comparable to RTQ-PCR measurements on intact RNA when simulating the RNA-species and tissue-specific degradation kinetic.

Cervical tissue collection methods for RNA preservation: comparison of snap-frozen, ethanol-fixed, and RNAlater-fixation.

Promising molecular techniques may allow for testing of novel and complex hypotheses such as defining gene expression profiles in specific cells, tumors, or their microenvironments. For most large cancer epidemiologic and population-based studies, however, application of such promising techniques may not be possible owing to constraints of specimen preservation from paraffin-embedded tissues. Alternative methods would ideally preserve tissue morphology and not degrade DNA or RNA. We conducted a comparison of snap-freezing (freezing with liquid nitrogen), ethanol-fixation with low melt polyester wax embedding, and RNAlater-preservation techniques to determine which method was optimal for subsequent assessment of gene expression changes in cervical cancer. From each of 15 women with cancer and 30 without, we procured 3 pieces of cervical tissue and compared snap-freezing, ethanol-fixation, and RNAlater-preservation techniques. Despite slight loss in morphologic quality from snap-frozen cervix tissues, RNA quality was equivalent to or better than RNAlater-preserved tissues and significantly exceeded that from ethanol-fixed/polyester wax embedded tissue. In conclusion, despite the moderate logistical constraints in set-up that required either liquid nitrogen or dry ice on-site for snap-freezing tissue, the ease of downstream processing and consistent high quality RNA made it preferable to the other 2 methods.

An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma.

Uveal (ocular) melanoma is an aggressive cancer that often forms undetectable micrometastases before diagnosis of the primary tumor. These micrometastases later multiply to generate metastatic tumors that are resistant to therapy and are uniformly fatal. We have previously identified a gene expression profile derived from the primary tumor that is extremely accurate for identifying patients at high risk of metastatic disease. Development of a practical clinically feasible platform for analyzing this expression profile would benefit high-risk patients through intensified metastatic surveillance, earlier intervention for metastasis, and stratification for entry into clinical trials of adjuvant therapy. Here, we migrate the expression profile from a hybridization-based microarray platform to a robust, clinically practical, PCR-based 15-gene assay comprising 12 discriminating genes and three endogenous control genes. We analyze the technical performance of the assay in a prospective study of 609 tumor samples, including 421 samples sent from distant locations. We show that the assay can be performed accurately on fine needle aspirate biopsy samples, even when the quantity of RNA is below detectable limits. Preliminary outcome data from the prospective study affirm the prognostic accuracy of the assay. This prognostic assay provides an important addition to the armamentarium for managing patients with uveal melanoma, and it provides a proof of principle for the development of similar assays for other cancers.