Molecular Diagnostic Panel Research Articles

Molecular Subgroups of Glioblastoma- an Assessment by Immunohistochemical Markers.

Comprehensive molecular characterization of and novel therapeutic approaches to glioblastoma have been explored as a result of advancements in biotechnologies. In this study, we aimed to bring basic research discoveries closer to clinical practice and ultimately incorporate molecular classification into the routine histopathological evaluation of grade IV gliomas. Integrated results of genome-wide sequencing, transcriptomic and epigenomic analyses by The Cancer Genome Atlas Network defined the classic, proneural, neural and mesenchymal subtypes of this tumor. In a retrospective cohort, we analyzed selected subgroup-defining molecular markers in formalin-fixed paraffin-embedded surgical specimens by immunohistochemistry. Quantitative and qualitative scores of marker expression were tested in hierarchical cluster analyses to evaluate segregations of the molecular subgroups, which then were correlated with clinical parameters including patients' age, gender and overall survival. Our study has confirmed the separation of molecular glioblastoma subgroups with clear trends regarding clinical correlations. Future analyses in a larger, prospective cohort using similar methods are expected to facilitate the development of a molecular diagnostic panel that may complement routine histological work up and support prognostication as well as treatment decisions in glioblastoma.

The First Fully Automated Molecular Diagnostic Panel for Meningitis and Encephalitis: How Well Does It Perform, and When Should It Be Used?

Rapid and accurate molecular diagnostic tests for the most common causes of infectious meningitis and encephalitis have the potential for high clinical impact. In this issue of the Journal of Clinical Microbiology, Leber et al. (J Clin Microbiol 54:2251-2261, 2016, http://dx.doi.org/10.1128/JCM.00730-16) report results from a large clinical study designed to prospectively assess the performance of the FilmArray meningitis/encephalitis panel compared to conventional methods.

Varicella zoster virus infection presenting as isolated diplopia: a case report

BackgroundInvolvement of trochlear nerve during Varicella Zoster Virus (VZV) Infection has been rarely described, and always in association with skin rash.Case presentationWe describe the case of a patient with VZV infection presenting as isolated diplopia due to fourth cranial nerve palsy. The diagnosis has been obtained through the application of a standardized molecular diagnostic panel, and diplopia resolved after specific antiviral and corticosteroid therapy.ConclusionThis case evidences that clinicians should be aware of atypical VZV infection, even in the absence of the typical skin rash.

A molecular diagnostic panel for thyroid cancer disease management.

5510 Background: In 2009, the American Thyroid Association revised its guidelines for patients with thyroid nodules and differentiated thyroid cancers, recommending BRAF, RAS, RET/PTC, and PAX8-PPARγ molecular testing in patients with indeterminate fine-needle aspirate (FNA) cytology. Alterations in any of these markers in thyroid nodules are strongly associated with malignancy. We studied the prevalence of these alterations in thyroid FNA specimens in order to establish a strategy to improve disease management. Methods: DNA and RNA were extracted from thyroid FNA specimens (N=149) and tested for BRAF mutations using allele-specific PCR (V600E and K601E); for RAS (H-, K-, N-) mutations using pyrosequencing (codons 12, 13, and 61); and for RET/PTC1 and RET/PTC3 rearrangements and PAX8-PPARγ translocations using RT-PCR. Results: Overall, 90 (60.4%) of the specimens had alterations in ≥1 of the 4 molecular markers (Table). BRAF V600E mutations (54.4%) were the most prevalent mutations in papillary thyroid cancer (PTC); no K601E mutations were found. RAS mutations were found in PTC, follicular adenoma (FA), and follicular thyroid cancer (FTC) specimens; half of these mutations involved N-RAS Q61. RET/PTC rearrangements were detected in 3.5% of PTC specimens and were evenly distributed between the 2 major subtypes, RET/PTC1 and RET/PTC3. PAX8/PPARγ translocations were detected in 18.8% of FTC but not in FA, probably due to small sample size. Out of 7 indeterminate thyroid nodules, 2 had BRAF mutations and 2 had RAS mutations. The presence of the 4 markers was generally mutually exclusive; only 1 PTC specimen had concurrent RAS and RET/PTC1 alterations. Conclusions: The prevalence of BRAF, RAS, RET/PTC, and PAX8/PPARγ alterations in thyroid cancer specimens highlights the potential for targeted therapeutic strategies. The mutually exclusive pattern of alterations also suggests a hierarchical screening strategy for samples with limited availability. [Table: see text]

Evidence from Multiplex Molecular Assays for Complex Multipathogen Interactions in Acute Respiratory Infections

While most diagnostic processes cease with the detection of the first relevant infectious agent, newer multiplexed molecular methods which provide simultaneous analysis of multiple agents may give a more accurate representation of the true pathogen spectrum in these samples. To examine this in the context of respiratory infections, acute-phase respiratory specimens submitted to our clinical diagnostic microbiology/virology laboratory for our routine VIRAP diagnosis protocol during the spring 2006 peak respiratory infection season were processed in parallel by analysis with Genaco (QiaPlex) ResPlex I and ResPlex II molecular diagnostic panels. A total of 1,742 specimens were examined for 21 relevant targets each. The resulting data reveal that multiple infections are frequent and provide evidence for complex interactions between different infectious agents. Statistically relevant association patterns (both positive and negative) were observed between particular pathogens. While some interactions we observed are substantiated by prior reports in the literature, several specific patterns do not appear to have been reported previously. In addition, we report preliminary clinical evidence which supports a hypothesis that these coinfections are medically relevant and that effective treatment for severe respiratory tract infections will increasingly require diagnosis of all involved pathogens, as opposed to single-pathogen reporting.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 86

At least 15 genetic loci and ten genes have been associated with the demyelinating form of Charcot‐Marie‐Tooth hereditary neuropathy and related disorders. As pathogenic mutations have been identified in more and more genes, the labor and expense of screening for such mutations has increased substantially. Using conventional methods such as direct sequencing, single‐strand conformation polymorphism (SSCP), or heteroduplex analysis, molecular diagnosis of CMT remains laborious due to the large number of exons to be screened. Some genes (MPZ, PMP22, EGR2, GJB1) are small and have to be analyzed in a relatively large number of patients while other rarer genes, such as periaxin (PRX, 19q13), are too large (7 exons encoding transcripts of 4.8–5.5 kilobases) to be easily included in a molecular diagnostic panel. Furthermore, the sensitivity of the more economical techniques (SSCP and heteroduplex analysis) is far from being optimal ( ). Denaturing High‐Performance Liquid Chromatography (DHPLC) is a newly developed method to scan DNA for mutations, which is capable of automated high‐throughput analysis that does not require modified PCR primers, specific reagent arrays or detection labels, or any sample pretreatment other than PCR. We have undertaken the mutational analysis of some genes associated with CMT1 and related neuropathies by using DHPLC. Optimal conditions have been determined for DHPLC analysis of MPZ, PMP22, GJB1 and PRX genes in an initial group of 30 patients with severe demyelinating neuropathy, negative for the common 17p11 duplication. The very high sensitivity of the method was demonstrated by analyzing a control group of patients with known mutations in the MPZ, PMP22 and GJB1 genes. The work is still in progress. Thus far, DHPLC has resolved all the mutations previously detected by sequencing and allowed the identification of a number of polymorphic variants in the PRX gene and a novel disease mutation in MPZ gene exon 4 in a patient with an intermediate (axonal‐demyelinating) form of CMT. Interestingly, this mutation had consistently appeared as homozygous at previous direct sequencing analysis. DHPLC clearly showed the heterozygous state of the mutation, thus demonstrating the power of this technique also in identifying sequencing artifact or mutation mosaicism, if any. Partially supported by a grant Ricerca Finalizzata Ministero della Sanità to FT.