Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR.

Activating mutations in EGFR predict benefit from tyrosine kinase inhibitor therapy for patients with advanced non-small cell lung cancer. Directing patients to appropriate therapy depends on accurate and timely EGFR assessment in the molecular pathology laboratory. This article describes the analytical design, performance characteristics, and clinical implementation of an assay for the rapid detection of EGFR L858R and exon 19 deletion mutations. A droplet digital polymerase chain reaction (ddPCR) assay was implemented with probe hydrolysis-dependent signal detection. A mutation-specific probe was used to detect EGFR L858R. A loss of signal design was used to detect EGFR exon 19 deletion mutations. Analytical sensitivity was dependent on DNA input and was as low as 0.01% variant allele fraction for the EGFR L858R assay and 0.1% variant allele fraction for the EGFR exon 19 deletion assay. Correlation of 20 clinical specimens tested by ddPCR and next generation sequencing showed 100% concordance. ddPCR showed 53% clinical sensitivity in the detection of EGFR mutations in plasma cell-free DNA from patients with lung cancer. The median clinical turnaround time was 5 days for ddPCR compared to 13 days for next generation sequencing. The findings show that ddPCR is an accurate and rapid method for detecting EGFR mutations in patients with non-small cell lung cancer.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/µL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing.

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

The Clinical Implications of Inconsistently Methylated Results from Glioblastoma MGMT Testing by Replicate Methylation-Specific PCR.

The methylation status of the promoter of the O6-methylguanine DNA methyltransferase gene (MGMT) is an established prognostic and predictive biomarker of glioblastoma (GBM). At the Center for Advanced Molecular Diagnostics, MGMT testing is performed by methylation-specific PCR with multiple replicates, leading to three types of reportable results: methylated, unmethylated, and inconsistently methylated. An inconsistently methylated result is reported when a methylated peak is seen in some but not all of the PCR replicates from a single DNA sample. To better understand the clinical implications of these results, we performed a retrospective review of all MGMT testing at our laboratory over a 5-year period, and correlated test results with outcome and specimen-quality data. This review yielded several novel findings. First, inconsistent MGMT methylation on replicate methylation-specific PCR is not uncommon, composes 12% (58/465) of our GBM results. Second, inconsistently methylated GBM cases are associated with relatively poor overall survival (more similar to unmethylated than to methylated cases). Third and interestingly, there appears to be a dose-response relationship between patient survival and the extent of methylation in inconsistently methylated GBMs. Finally, our analyses of specimen-quality data suggest that a combination of technical factors (eg, small samples) and tumor biology may explain inconsistent MGMT results on replicate methylation-specific PCR testing.

Pulmonary large cell carcinoma lacking squamous differentiation is clinicopathologically indistinguishable from solid-subtype adenocarcinoma.

CONTEXT: Pulmonary large cell carcinoma (LCC) includes tumors not readily diagnosed as adenocarcinoma (ADC) or squamous cell carcinoma on morphologic grounds, without regard to immunophenotype, according to the World Health Organization (WHO). This ambiguous designation may cause confusion over selection of mutation testing and directed therapies. Several groups have proposed the use of immunohistochemistry (IHC) to recategorize LCC as ADC or squamous cell carcinoma; however, it remains unclear if strictly defined LCCs are a clinicopathologically distinct lung tumor subset. OBJECTIVE: To compare the pathologic, molecular, and clinical features of 2 morphologically similar tumors: solid-subtype ADC and LCC. DESIGN: Tumors were included on the basis of solid growth pattern; tumors with squamous or neuroendocrine differentiation were excluded. Solid ADC (n = 42) and LCC (n = 57) were diagnosed by using WHO criteria (5 intracellular mucin droplets in ≥2 high-power fields for solid ADC) and tested for KRAS, EGFR, and ALK alterations. RESULTS: Both solid ADC and LCC groups were dominated by tumors with "undifferentiated"-type morphology and both had a high frequency of thyroid transcription factor 1 expression. KRAS was mutated in 38% of solid ADCs versus 43% of LCCs (P = .62). One ALK-rearranged and 1 EGFR-mutated tumor were detected in the solid ADC and LCC groups, respectively. There were no significant differences in clinical features or outcomes; the prevalence of smoking in both groups was greater than 95%. CONCLUSIONS: Other than a paucity of intracellular mucin, LCC lacking squamous or neuroendocrine differentiation is indistinguishable from solid-subtype ADC. We propose the reclassification of these tumors as mucin-poor solid adenocarcinomas.