Role of Prescription Practice in Ensuring Patient Safety in the Primary Health-Care Settings in India.

"Completeness (a measure of adequacy)" and the "appropriateness (a measure of the quality of care)" are two dimensions of good prescription practice. The study assessed the prescription practices at the primary health centers (PHCs); to demonstrate the effect of individual and system-level factors, on adequacy and appropriateness of prescription practices, with special reference to e-prescription over manual prescription given the rising acceptance of teleconsultation in health care. A total of 600 manual and 1000 e-prescriptions were randomized using a probability-proportional-to-size sampling method to distribute/allocate samples across manual and e-prescriptions. Findings revealed that while adequacy and appropriateness of prescriptions depend on individual training and clinical practice; adequacy of prescription, especially the manual was compromised by systemic factors, such as nonavailability of space in a prescription, forcing doctors to prioritize documentation of diagnosis, advising tests, and prescribing medicines, over other details (chief complaints and examination findings).

Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology.

Screening for genomic sequence variants in genes of predictive and prognostic significance is an integral part of precision medicine. Next-generation sequencing (NGS) technologies are progressively becoming platforms of choice to facilitate this, owing to their massively parallel sequencing capability, which can be used to simultaneously screen multiple markers in multiple samples for a variety of variants (single nucleotide and multi nucleotide variants, insertions and deletions, gene copy number variations, and fusions). A crucial step in the workflow of targeted NGS is the enrichment of the genomic regions of interest to be sequenced, against the whole genomic background. This ensures that the NGS effort is focused to predominantly screen target regions of interest with minimal off-target sequencing, making it more accurate and economical. Polymerase chain reaction-based (PCR, or amplicon-based) and hybridization capture-based methodologies are the two prominent approaches employed for target enrichment. This review summarizes the basic principles of target enrichment utilized by these methods, their multiple variations that have evolved over time, automation approaches, overall comparison of their advantages and drawbacks, and commercially available choices for these methodologies.

Next-Generation Sequencing in High-Sensitive Detection of Mutations in Tumors: Challenges, Advances, and Applications.

Next-generation sequencing (NGS) technologies have come of age as preferred technologies for screening of genomic variants of pathologic and therapeutic potential. Because of their capability for high-throughput and massively parallel sequencing, they can screen for a variety of genomic changes in multiple samples simultaneously. This has made them platforms of choice for clinical testing of solid tumors and hematological malignancies. Consequently, they are increasingly replacing conventional technologies, such as Sanger sequencing and pyrosequencing, expression arrays, real-time PCR, and fluorescence in situ hybridization methods, for routine molecular testing of tumors. However, one limitation of routinely used NGS technologies is the inability to detect low-level genomic variants with high accuracy. This can be attributed to the frequent occurrence of low-level sequencing errors and artifacts in NGS workflow that need specialized approaches to be identified and eliminated. This review focuses on the origins and nature of these artifacts and recent improvements in the NGS technologies to overcome them to facilitate accurate high-sensitive detection of low-level mutations. Potential applications of high-sensitive NGS in oncology and comparisons with non-NGS technologies of similar capabilities are also summarized.

Hybrid oncocytic/chromophobe renal tumors are molecularly distinct from oncocytoma and chromophobe renal cell carcinoma.

Hybrid oncocytic/chromophobe tumor (HOCT) of the kidney represents a poorly understood clinicopathologic entity with pathologic features that overlap between benign renal oncocytoma (RO) and malignant chromophobe renal cell carcinoma (ChRCC). Consequently, characterization of HOCT and its separation from the foregoing entities are clinically important. The aim of this study was to describe the pathologic and molecular features of HOCT and to compare them with those of RO and ChRCC. We retrospectively identified a cohort of 73 cases with renal oncocytic tumors (19 RO, 27 HOCT, and 27 ChRCC) for whom clinical follow-up data were available by 2 tertiary care hospitals. All cases were sporadic except for 2 HOCTs that were associated with Birt-Hogg-Dubé syndrome. Lesional tissues were retrieved for molecular analysis. We performed targeted gene sequencing of all exons of 261 cancer related genes on a subset of HOCT samples (n = 16). Gene expression profiling of a customized codeset was conducted on 19 RO, 24 HOCT, and 27 ChRCC samples. Clinicopathologic characteristics as well as DNA copy number alterations, mutational and transcriptional features of HOCT derived from sequencing and expression profiling data are described and compared to those in RO and ChRCC. HOCTs were more frequently multifocal and did not exhibit mutations in genes that are recurrently mutated in RO or ChRCC but showed copy number alterations primarily involving losses in chromosomes 1 and X/Y. The mRNA transcript data show that HOCT can be separated from RO and ChRCC. Hence, HOCT appears to represent a distinct renal tumor entity with genomic features that are intermediate between those of RO and ChRCC.

Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era.

ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease.

Challenges in next generation sequencing analysis of somatic mutations in transplant patients.

Analysis of somatic mutations in solid tumors and hematologic malignancies using targeted next generation sequencing (NGS)-based assays has become part of routine oncology practice as well as clinical trials. The use of paired tumor-normal DNA samples increases confidence of somatic calls. NGS assays that utilize unique patient identifiers (SNP IDs) allow further comparison of samples within a run or paired tumor/normal samples. The sources of germline DNA include peripheral blood (PB) and formalin-fixed paraffin-embedded tissue (FFPE). However, the source of normal can be problematic, especially in transplant setting. Herein, we report two cases of NGS-based molecular testing in a patient with mycosis fungoides treated with stem cell transplant [SCT] (Pt1) and a patient with lung adenocarcinoma who previously had acute leukemia cured by SCT. These cases highlight the importance of selecting an appropriate normal sample for excluding germline polymorphisms during somatic mutation testing. Initial analyses that included concurrent PB sample failed to filter known germline polymorphisms. Repeat analyses using pre-transplant PB/bone marrow allowed for the successful subtraction of germline variants. Somatic mutations in PTEN and ERBB4 (Pt1) and CDKN2A, KRAS, KDR, and TP53 (Pt2) were reported with confidence. Selection of an appropriate source of germline DNA for NGS-based somatic mutation testing for patients with SCT transplant can be challenging. Particular attention to the clinical history is crucial for accurate interpretation and reporting.

MET amplification assessed using optimized FISH reporting criteria predicts early distant metastasis in patients with non-small cell lung cancer.

To investigate the prognostic impact of MET copy number (MET-CN) in patients with non-small cell lung cancer (NSCLC), we retrospectively reviewed clinical and pathologic data of NSCLC patients whose tumors were assessed for MET-CN using fluorescence in situ hybridization (FISH). We correlated MET-CN status with patient overall survival (OS) and optimized MET-FISH reporting criteria. The study group included 384 patients with NSCLC of which 88% were adenocarcinoma and 55.7% of patients had distant metastases. There were 170 patients with stages I-III and 214 patients with stage IV disease. Based on the MET-CN and MET/CEP7 ratio the patients were classified into 3 categories: MET-amplification (METamp): MET/CEP7 ≥ 2 or MET-CN ≥ 5; MET-CN-gain (METcng): MET-CN ≥ 4 to < 5; and MET-negative (METneg): MET-CN < 4. METamp was associated with high fatality (P=.036) and stage IV tumors (P=.038). In patients with stages I-III NSCLC, patients in the METamp category had the shortest OS (P=.015) and more often developed distant metastases within 1 year (P=.004). In patients with stage IV tumors, METamp did not further impact the OS. Patients in the METcng category had the longest OS (P=.053). Multivariate analysis confirmed METamp to be an independent high-risk factor (HR 3.26; P=.026) and predicted earlier progression to distant metastasis (HR 4.86; P=.001). In conclusion, we suggest that the MET-FISH criteria presented optimizes risk stratification by defining 3 categories of NSCLC patients. METamp is an independent risk factor predicting early distant metastasis and patients with METcng could represent a lower-risk group.

Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors.

A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton. A subset of these samples with DNA availability was tested by ddPCR and UltraSEEK MassARRAY for mutation detection in 5 genes (IDH1, PIK3CA, KRAS, BRAF, and NRAS). Sixty one of 104 expected tissue mutations and 6 additional mutations not present in the tissue were detected in cfDNA. ddPCR and MassARRAY showed 83% and 77% concordance with NGS for mutation detection with 100% and 79% sensitivity, respectively. The median OS of patients with lower cfDNA yield (74 vs 50 months; P < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; p < 0.04) was significantly longer than in patients with higher cfDNA yield and positive for mutations. A limit-of-detection of 0.1% was demonstrated for ddPCR and MassARRAY platforms using a serially diluted positive cfDNA sample. The MassARRAY and ddPCR systems enable fast and cost-effective genotyping for a targeted set of mutations and can be used for single gene testing to guide response to chemotherapy or for orthogonal validation of NGS results.

Versatile ion S5XL sequencer for targeted next generation sequencing of solid tumors in a clinical laboratory.

BACKGROUND: Next generation sequencing based tumor tissue genotyping involves complex workflow and a relatively longer turnaround time. Semiconductor based next generation platforms varied from low throughput Ion PGM to high throughput Ion Proton and Ion S5XL sequencer. In this study, we compared Ion PGM and Ion Proton, with a new Ion S5XL NGS system for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing performance in a clinical laboratory. METHODS: Eighteen solid tumor samples positive for various mutations as detected previously by Ion PGM and Ion Proton were selected for study. Libraries were prepared using DNA (range10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.0 for comprehensive cancer (CCP), oncomine comprehensive cancer (OCP) and cancer hotspot panel v2 (CHPv2) panel as per manufacturer's instructions. The CHPv2 were sequenced using Ion PGM whereas CCP and OCP were sequenced using Ion Proton respectively. All the three libraries were further sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data analysis was performed using Torrent Suite 4.6 software on board S5XL and Ion Reporter. A limit of detection and reproducibility studies was performed using serially diluted DLD1 cell line. RESULTS: A total of 241 variant calls (235 single nucleotide variants and 6 indels) expected in the studied cohort were successfully detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was reduced from 4.5 to 2.5 hours with output range of 3-5 GB (S530) and 8-9.3Gb (S540). Data analysis time for the Ion S5XL is faster 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as compared to the Ion PGM (3.5-5 h) and Ion Proton (8h). A limit detection of 5% allelic frequency was established along with high inter-run reproducibility. CONCLUSION: Ion S5XL system simplified workflow in a clinical laboratory, was feasible for running smaller and larger panels on the same instrument, had a shorter turnaround time, and showed good concordance for variant calls with similar sensitivity and reproducibility as the Ion PGM and Proton.

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid.

Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRAS p.Q61H and EZH2 p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.

Validation of quantitative PCR-based assays for detection of gene copy number aberrations in formalin-fixed, paraffin embedded solid tumor samples.

Gene copy number changes are important somatic alterations in cancers. A number of high throughput methods, such as next generation sequencing, are capable of detecting copy number aberrations, but their use can be challenging and cost prohibitive for screening a small number of markers. Furthermore, detection of CNAs by high throughput platforms needs confirmation by an orthogonal technique, especially in cases with low level CNAs. Here, we have validated TaqMan based quantitative PCR (qPCR) assays to detect CNAs in genes of high clinical importance in formalin-fixed, paraffin-embedded (FFPE) samples. A cohort of 22 tumors of various types that harbor 67 CNAs in 13 genes was assessed. The abnormalities in these tumors were detected by using a NGS-based 50 gene hotspot panel on Ion Torrent PGM and molecular inversion probe (MIP) array. The CNAs included ERBB2 (n = 6), PDGFRA (n = 6), KIT (n = 7), NRAS (n = 3), PIK3CA (n = 6), MYC (n = 7), MET (n = 4), FLT3 (n = 6), FGFR3 (n = 3), FGFR2 (n = 3), EGFR (n = 7), KRAS (n = 6) and FGFR1 (n = 5). Different amounts of input DNA were tested and 5 ng FFPE DNA was found to be adequate without limiting detection sensitivity. All 22 (100%) positive tumor samples revealed by MIP array were confirmed by real time qPCR and 17 of 22 (77.2%) samples tested by NGS were confirmed. The limit of detection of the qPCR assay was determined by serial dilution of SKBR3 cell line DNA (with amplified ERBB2) and showed an ability to detect 3 copies consistently up to 0.75% dilution. The ability to use low input of FFPE DNA, high sensitivity, and short turnaround time makes qPCR a valuable and economically viable platform for detecting single gene CNAs as well as for confirmation of CNAs detected by high throughput screening assays.

Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial.

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

Concurrent fine needle aspirations and core needle biopsies: a comparative study of substrates for next-generation sequencing in solid organ malignancies.

Minimally invasive procedures, such as fine needle aspiration and core needle biopsy, are commonly used for the diagnosis in solid organ malignancies. In the era of targeted therapy, it is crucial for molecular testing to be performed on these limited volume specimens. Although several recent studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to whether core needle biopsy specimens are more reliable than fine needle aspiration for molecular studies. In this study, we reviewed concurrently acquired fine needle aspiration and core needle biopsy samples (n=24), and compared overall cellularity, tumor fraction, and the results of next-generation sequencing. All somatic mutations detected in core needle biopsy samples were also detected in fine needle aspiration samples. The estimated tumor fraction was significantly higher in fine needle aspiration smears than core needle biopsy samples (P=0.003), whereas the overall DNA yield from smears was significantly lower than that obtained from the core needle biopsy specimens (P=0.01). The normalized average amplicon coverage for the genes analyzed was significantly higher in cytology smears than paired core needle biopsy samples, with lower numbers of failed amplicons and higher overall mutation allelic frequencies seen in the former. We further evaluated 100 malignant fine needle aspiration and core needle biopsy samples, acquired concurrently, for overall cellularity and tumor fraction. Overall cellularity and tumor fraction of fine needle aspiration samples was significantly higher than concurrently acquired core needle biopsy samples (P<0.001). In conclusion, we show that fine needle aspiration samples frequently provide better cellularity, higher tumor fraction, and superior sequencing metrics than concurrently acquired core needle biopsy samples. Cytologic specimens, therefore, should be better integrated into routine molecular diagnostics workflow to maximize limited tissues for clinically relevant genomic testing.

Genome-wide copy number aberrations and HER2 and FGFR1 alterations in primary breast cancer by molecular inversion probe microarray.

Breast cancer remains the second leading cause of cancer-related death in women despite stratification based on standard hormonal receptor (HR) and HER2 testing. Additional prognostic markers are needed to improve breast cancer treatment. Chromothripsis, a catastrophic genome rearrangement, has been described recently in various cancer genomes and affects cancer progression and prognosis. However, little is known about chromothripsis in breast cancer. To identify novel prognostic biomarkers in breast cancer, we used molecular inversion probe (MIP) microarray to explore genome-wide copy number aberrations (CNA) and breast cancer-related gene alterations in DNA extracted from formalin-fixed paraffin-embedded tissue. We examined 42 primary breast cancers with known HR and HER2 status assessed via immunohistochemistry and FISH and analyzed MIP microarray results for correlation with standard tests and survival outcomes. Global genome-wide CNA ranged from 0.2% to 65.7%. Chromothripsis-like patterns were observed in 23/38 (61%) cases and were more prevalent in cases with ≥10% CNA (20/26, 77%) than in cases with <10% CNA (3/12, 25%; p<0.01). Most frequently involved chromosomal segment was 17q12-q21, the HER2 locus. Chromothripsis-like patterns involving 17q12 were observed in 8/19 (42%) of HER2-amplified tumors but not in any of the tumors without HER2 amplification (0/19; p<0.01). HER2 amplification detected by MIP microarray was 95% concordant with conventional testing (39/41). Interestingly, 21% of patients (9/42) had fibroblast growth factor receptor 1 (FGFR1)amplification and had a 460% higher risk for mortality than those without FGFR1 amplification (p<0.01). In summary, MIP microarray provided a robust assessment of genomic CNA of breast cancer.

A Targeted High-Throughput Next-Generation Sequencing Panel for Clinical Screening of Mutations, Gene Amplifications, and Fusions in Solid Tumors.

Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumors. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumor samples representing 13 tumor types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay-Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumors.

Real-Time Genomic Characterization Utilizing Circulating Cell-Free DNA in Patients with Anaplastic Thyroid Carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is an aggressive disease that requires rapid diagnosis and multimodality treatment. Recent advances in targeted therapeutics have provided ATC patients with previously unavailable treatment options, which may improve clinical outcomes in the coming years. Continued development of high-throughput next-generation sequencing provides clinicians with an unparalleled ability to characterize the genomic background of tumors in order to guide treatment selection and clinical trial enrollment. METHODS: Twenty-three patients with ATC treated at the University of Texas MD Anderson Cancer Center between August 2015 and April 2016 were evaluated. All patients underwent next-generation sequencing using an institutional tissue-based DNA platform (50 genes) and a commercially available cell-free circulating DNA (cfDNA) platform (70 genes). RESULTS: Sequencing data were successfully obtained for both platforms on all patients. The most commonly mutated genes noted on both platforms were TP53 (15/23; 65%) and BRAF (11/23; 48%). Concordance between the tumor and cfDNA data was high for BRAF, PIK3CA, NRAS, and PTEN and moderate for TP53. Concordance was highest in patients who underwent dual-platform sequencing prior to initiation of definitive treatment, and lowest in patients who underwent cfDNA analysis following treatment. Nineteen patients had treatment at the University of Texas MD Anderson Cancer Center following cfDNA sequencing. One patient was observed, and three patients opted for hospice. At the time of last contact, 15/23 (65%) patients were alive. CONCLUSIONS: Next-generation sequencing platforms offer clinicians an opportunity to identify targetable oncogenic events in ATC. To the authors' knowledge, this is the largest sequential cohort of ATC patients who have undergone targeted genomic profiling. Based on these data, utilization of both tumor-based and cfDNA analysis in the context of clinical-trial development and application is recommended. Integration of these or similar platforms in clinical-trial implementation may have the potential to transform clinical outcomes for patients with ATC.

Comprehensive Screening of Gene Copy Number Aberrations in Formalin-Fixed, Paraffin-Embedded Solid Tumors Using Molecular Inversion Probe-Based Single-Nucleotide Polymorphism Array.

Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes. A cohort of 76 solid tumors of various types and tumor cellularity (20% to 100%), and four cancer cell lines were used. These harbored CNAs in clinically important genes (ERBB2, EGFR, FGFR1, KRAS, MYC) as detected by orthogonal techniques like next-generation sequencing or fluorescence in situ hybridization. Results of the MIP array were concordant with results from orthogonal techniques, and also provided additional information regarding the allelic nature of the CNAs. Limit-of-detection and assay reproducibility studies showed a high degree of sensitivity and reproducibility of detection, respectively. FFPE compatibility, ability to detect CNAs with high sensitivity, accuracy, and provide valuable information such as loss of heterozygosity along with relatively short turnaround times makes the MIP array a desirable clinical platform for routine screening of solid tumors in a clinical laboratory.

Next-generation sequencing identifies high frequency of mutations in potentially clinically actionable genes in sebaceous carcinoma.

Sebaceous carcinoma (SC) is a rare but aggressive malignancy with frequent recurrence and metastases. Surgery is the mainstay of therapy, but effective systemic therapies are lacking because the molecular alterations driving SC remain poorly understood. To identify these, we performed whole-exome next-generation sequencing of 409 cancer-associated genes on 27 SCs (18 primary/locally recurrent ocular, 5 paired metastatic ocular, and 4 primary extraocular) from 20 patients. In ocular SC, we identified 139 non-synonymous somatic mutations (median/lesion 3; range 0-23). Twenty-five of 139 mutations (18%) occurred in potentially clinically actionable genes in 6 of 16 patients. The most common mutations were mutations in TP53 (n = 9), RB1 (n = 6), PIK3CA (n = 2), PTEN (n = 2), ERBB2 (n = 2), and NF1 (n = 2). TP53 and RB1 mutations were restricted to ocular SC and correlated with aberrant TP53 and RB protein expression. Systematic pathway analyses demonstrated convergence of these mutations to activation of the PI3K signalling cascade, and PI3K pathway activation was confirmed in tumours with PTEN and/or PIK3CA mutations. Considerable inter-tumoural heterogeneity was observed between paired primary and metastatic ocular SCs. In primary extraocular SC, we identified 77 non-synonymous somatic mutations (median/lesion 22.5; range 3-29). This overall higher mutational load was attributed to a microsatellite instability phenotype in three of four patients and somatically acquired mutations in mismatch repair genes in two of four patients. Eighteen of 77 mutations (23%) were in potentially clinically actionable genes in three of four patients, including BTK, FGFR2, PDGFRB, HRAS, and NF1 mutations. Identification of potentially clinically actionable mutations in 9 of 20 SC patients (45%) underscores the importance of next-generation sequencing to expand the spectrum of genotype-matched targeted therapies. Frequent activation of PI3K signalling pathways provides a strong rationale for application of mTOR inhibitors in the management of this disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of Factors Affecting the Success of Next-Generation Sequencing Testing in Solid Tumors.

OBJECTIVES: Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results. METHODS: We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance. RESULTS: Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm(2) (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid-based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (<10 mm(2)) samples (from 76% to 94%) as well as cytology samples (from 58% to 87%). CONCLUSIONS: Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures.

Genotyping of Frequent Mutations in Solid Tumors by PCR-Based Single-Base Extension and MassARRAY Analysis.

Over the last decade, cancer genome sequencing has revealed in detail the genomic landscapes of an increasing number of common solid human tumors. This has greatly impacted the clinical care of cancer patients based on the fact that many of these tumors exhibit activating mutations in driver genes that are prone to target therapy, largely impacting cancer management strategies. Genomic heterogeneity of tumors is becoming an increasingly recognized phenomenon relevant to genome-based medicine. Because a large number tumors may display several mutations at the same time, multiplexing has become the preferred approach to reveal the mutational landscape in patient samples and to better design a targeted approach to their illness.