As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop ...and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.
Introduction:
In metastatic colorectal cancer (mCRC), RAS mutations impart inferior survival and resistance to anti-epidermal growth factor receptor (EGFR) antibodies. KRAS G12C inhibitors have been ...developed and we evaluated how KRAS G12C differs from other RAS mutations.
Patients and Methods:
This retrospective review evaluated patients in British Columbia, Canada with mCRC and RAS testing performed between 1 January 2016 and 31 December 2018. Sequencing information from The Cancer Genome Analysis (TCGA) was also obtained and analysed.
Results:
Age at diagnosis, sex, anatomic location and stage at diagnosis did not differ by RAS mutation type. Progression free survival on first chemotherapy for patients with metastatic KRAS G12C tumours was 11 months. Median overall survival did not differ by RAS mutation type but was worse for both KRAS G12C (27 months) and non-G12C alterations (29 months) than wildtype (43 months) (p = 0.01). Within the TCGA, there was no differential gene expression between KRAS G12C and other RAS mutations. However, eight genes with copy number differences between the G12C and non-G12C RAS mutant groups were identified after adjusting for multiple comparisons (FITM2, PDRG1, POFUT1, ERGIC3, EDEM2, PIGU, MANBAL and PXMP4). We also noted that other RAS mutant mCRCs had a higher tumour mutation burden than those with KRAS G12C mutations (median 3.05 vs 2.06 muts/Mb, p = 4.2e–3) and that KRAS G12C/other RAS had differing consensus molecular subtype distribution from wildtype colorectal cancer (CRC) (p < 0.0001) but not each other (p = 0.14).
Conclusion:
KRAS G12C tumours have similar clinical presentation to other RAS mutant tumours, however, are associated with differential copy number alterations.
Assessing Limit of Detection in Clinical Sequencing Starks, Elizabeth R.; Swanson, Lucas; Docking, T. Roderick ...
The Journal of molecular diagnostics : JMD,
April 2021, 2021-Apr, 2021-04-00, 20210401, Letnik:
23, Številka:
4
Journal Article
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Clinical reporting of solid tumor sequencing requires reliable assessment of the accuracy and reproducibility of each assay. Somatic mutation variant allele fractions may be below 10% in many samples ...due to sample heterogeneity, tumor clonality, and/or sample degradation in fixatives such as formalin. The toolkits available to the clinical sequencing community for correlating assay design parameters with assay sensitivity remain limited, and large-scale empirical assessments are often relied upon due to the lack of clear theoretical grounding. To address this uncertainty, a theoretical model was developed for predicting the expected variant calling sensitivity for a given library complexity and sequencing depth. Binomial models were found to be appropriate when assay sensitivity was only limited by library complexity or sequencing depth, but functional scaling for library complexity was necessary when both library complexity and sequencing depth were co-limiting. This model was empirically validated with sequencing experiments by using a series of DNA input amounts and sequencing depths. Based on these findings, a workflow is proposed for determining the limiting factors to sensitivity in different assay designs, and the formulas for these scenarios are presented. The approach described here provides designers of clinical assays with the methods to theoretically predict assay design outcomes a priori, potentially reducing burden in clinical tumor assay design and validation efforts.
Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) ...are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF.
A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF.
We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy.
Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.
We compared clinical validity of two non-invasive prenatal screening (NIPS) methods for fetal trisomies 13, 18, 21, and monosomy X. We recruited prospectively 2203 women at high risk of fetal ...aneuploidy and 1807 at baseline risk. Three-hundred and twenty-nine euploid samples were randomly removed. The remaining 1933 high risk and 1660 baseline-risk plasma aliquots were assigned randomly between four laboratories and tested with two index NIPS tests, blind to maternal variables and pregnancy outcomes. The two index tests used massively parallel shotgun sequencing (semiconductor-based and optical-based). The reference standard for all fetuses was invasive cytogenetic analysis or clinical examination at birth and postnatal follow-up. For each chromosome of interest, chromosomal ratios were calculated (number of reads for chromosome/total number of reads). Euploid samples' mean chromosomal ratio coefficients of variation were 0.48 (T21), 0.34 (T18), and 0.31 (T13). According to the reference standard, there were 155 cases of T21, 49 T18, 8 T13 and 22 45,X. Using a fetal fraction ≥4% to call results and a chromosomal ratio z-score of ≥3 to report a positive result, detection rates (DR), and false positive rates (FPR) were not statistically different between platforms: mean DR 99% (T21), 100%(T18, T13); 79%(45,X); FPR < 0.3% for T21, T18, T13, and <0.6% for 45,X. Both methods' negative predictive values in high-risk pregnancies were >99.8%, except for 45,X(>99.6%). Threshold analysis in high-risk pregnancies with different fetal fractions and z-score cut-offs suggested that a z-score cutoff to 3.5 for positive results improved test accuracy. Both sequencing platforms showed equivalent and excellent clinical validity.
Non-invasive prenatal aneuploidy testing (NIPT) by next-generation sequencing of circulating cell-free DNA in maternal plasma relies on chromosomal ratio (chrratio) measurements to detect aneuploid ...values that depart from euploid ratios. Diagnostic performances are known to depend on the fraction of fetal DNA (FF) present in maternal plasma, although how this translates into specific quantitative changes in specificity/positive predictive values and which other variables might also be important is not well understood.
To explore this issue, theoretical relationships between FF and various measures of diagnostic performances were assessed for a range of parameter values. Empirical data from three NIPT assays were then used to validate theoretical calculations.
For a given positivity threshold, dramatic changes in specificity and positive predictive values (PPV) as a function of both FF and the coefficient of variation (CV) of the chrratio measurement were observed. Theoretically predicted and observed chrratio z-scores agreed closely, confirming the determinant impact of small changes in both FF and chrratio CV.
Evaluation of NIPT assay performances therefore requires knowledge of the FF distribution in the population in which the test is intended to be used and, in particular, of the precise value of the assay chrratio CV for each chromosome or genomic region of interest. Laboratories offering NIPT testing should carefully measure these parameters to ensure test reliability and clinical usefulness in interpreting individual patients' results.
•NIPT assays performances are determined by fetal fraction and chromosomal ratio CV.•Knowledge of these parameters allow to predict sample z-score values.•Positive predictive values may vary substantially with sample fetal fractions.•Global performance may not meaningfully reflect individual sample likelihoods.
Sample Tracking Using Unique Sequence Controls Moore, Richard A.; Zeng, Thomas; Docking, T. Roderick ...
The Journal of molecular diagnostics : JMD,
February 2020, 2020-02-00, 20200201, Letnik:
22, Številka:
2
Journal Article
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Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical ...setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.
Next-generation sequencing assays are capable of identifying cancer patients eligible for targeted therapies and can also detect germline variants associated with increased cancer susceptibility. ...However, these capabilities have yet to be routinely harmonized in a single assay because of challenges with accurately identifying germline variants from tumor-only data. We have developed the Oncology and Hereditary Cancer Program targeted capture panel, which uses tumor tissue to simultaneously screen for both clinically actionable solid tumor variants and germline variants across 45 genes. Validation using 14 tumor specimens, composed of patient samples and cell lines analyzed in triplicate, demonstrated high coverage with sensitive and specific identification of single-nucleotide variants and small insertions and deletions. Average coverage across all targets remained >2000× in 198 additional patient tumor samples. Analysis of 55 formalin-fixed, paraffin-embedded tumor samples for the detection of known germline variants within a subset of cancer-predisposition genes, including one multiexon deletion, yielded a 100% detection rate, demonstrating that germline variants can be reliably detected in tumor samples using a single panel. Combining targetable somatic and actionable germline variants into a single tumor tissue assay represents a streamlined approach that can inform treatment for patients with advanced cancers as well as identify those with potential germline variants who are eligible for confirmatory testing, but would not otherwise have been identified.
Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts ...that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. Several parameters relating to sequencing quality and variant calling were examined and quantified in tumor and normal colon epithelial tissues. We identified an alternative fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathologic review.
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