Lung adenocarcinoma with EGFR L858R-K860I and L858R-L861F doublet mutations from which the L858R mutation is undetectable through the cobas EGFR mutation test v2.

In East Asia, epidermal growth receptor factor (EGFR) mutations are the most prevalent and important biomarkers for treating patients with advanced lung cancer. However, as L858R doublet mutations are rare, commercially available EGFR tests may yield false-negative results. To determine whether the L858R mutation of the L858R-K860I and L858R-L861F doublet mutations could be identified using different types of EGFR detection tests and to describe the clinical response of patients with lung cancer with L858R doublet mutations to EGFR tyrosine kinase inhibitors (TKI). Information and samples from four patients with L858R doublet mutations, including three with L858R-K860I and one with L858R-L861F, were retrospectively collected from the archives of our department. For each case, the clinical response to EGFR-TKI was retrieved from the medical records. Archived formalin-fixed paraffin-embedded blocks were subjected to Sanger sequencing, the cobas and Idylla EGFR tests, the IntelliPlex-LCP-DNA assay, and AmoyDx PLC panel. L858R mutations were all detected by Sanger sequencing and the Idylla EGFR test but missed by the cobas assay. The AmoyDx PLC detected L858R only in cases with L858R-K860I while the IntelliPlex-LCP-DNA assay detected L858R in the case with L858R-L861F. Additionally, three of the patients, who had measurable tumors, showed partial responses to afatinib and osimertinib. The L858R mutation associated with L858R-K860I and L858R-L861F doublet mutations could be detected using Idylla but not cobas EGFR tests. Using next-generation sequencing analysis should be considered after initial negative reports from the cobas test, because patients with L858R doublet mutations may benefit from EGFR-TKIs.

The association of EGFR amplification with aberrant exon 20 insertion report using the cobas EGFR Mutation Test v2.

Determining the exact type of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation in lung cancer has become important. We found that not all ex20ins mutations reported by cobas EGFR test v2 could be validated by Sanger sequencing even using surgical specimens with high tumor contents. This study aimed to validate the ex20ins results reported by the cobas test and to determine whether there were clinicopathological factors associated with aberrant cobas ex20ins report. In total, 123 cobas-reported cases with ex20ins were retrospectively collected and validated by Sanger sequencing and Idylla assay. Clinicopathological features between ex20ins cobas+/Sanger+ group (n = 71) and cobas+/Sanger- group (n = 52) were compared. The Idylla assay detected ex20ins in 82.6% of cobas+/Sanger+ cases but only in 4.9% of cobas+/Sanger- cases. The cobas+/Sanger- group was significantly associated with higher tumor contents, poorly differentiated patterns, tumor necrosis, and a lower internal control cycle threshold value reported by the Idylla which suggesting the presence of increased EGFR gene copy numbers. EGFR fluorescence in situ hybridization (FISH) revealed the majority of cobas+/Sanger- group had EGFR high copy number gain (16%) or amplification (76%) according to the Colorado criteria. Among cases reported to have concomitant classic EGFR and ex20ins mutations by the cobas, the classic EGFR mutations were all detected by Sanger sequencing and Idylla, while the ex20ins mutations were undetected by Sanger sequencing (0%) or rarely reported by Idylla assay (3%). FISH revealed high EGFR copy number gain (17.9%) and amplification (79.5%) in cases reported having concomitant classic EGFR and ex20ins mutations by the cobas. This study demonstrated an unusually high frequency of EGFR amplification in cases with aberrant cobas ex20ins report which could not be validated by Sanger sequencing or Idylla assay. Ex20ins reported by the cobas test should be validated using other methods especially those reported having concomitant ex20ins and classic EGFR mutations.

Droplet digital polymerase chain reaction detection of KRAS mutations in pancreatic FNA samples: Technical and practical aspects for routine clinical implementation.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is associated with a 5-year survival rate of less than 6%, and current treatments have limited efficacy. The diagnosis of PDAC is mainly based on a cytologic analysis of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) samples. However, the collected specimens may prove noncontributory in a significant number of cases, delaying patient management and treatment. The combination of EUS-FNA sample examination and KRAS mutation detection can improve the sensitivity for diagnosis. In this context, the material used for molecular analysis may condition performance. METHODS: The authors prospectively compared the performance of cytologic analysis combined with a KRAS droplet digital polymerase chain reaction (ddPCR) assay for PDAC diagnosis using either conventional formalin-fixed, paraffin-embedded cytologic samples or needle-rinsing fluids. RESULTS: Molecular testing of formalin-fixed, paraffin-embedded cytologic samples was easier to set up, but the authors observed that the treatment of preanalytic samples, in particular the fixation process, drastically reduced ddPCR sensitivity, increasing the risk of false-negative results. Conversely, the analysis of dedicated, fresh needle-rinsing fluid samples appeared to be ideal for ddPCR analysis; it had greater sensitivity and was easily to implement in clinical use. In particular, fluid collection by the endoscopist, transportation to the laboratory, and subsequent freezing did not affect DNA quantity or quality. Moreover, the addition of KRAS mutation detection to cytologic examination improved diagnosis performance, regardless of the source of the sample. CONCLUSIONS: Considering all of these aspects, the authors propose the use of an integrated flowchart for the KRAS molecular testing of EUS-FNA samples in clinical routine.

Rapid and reliable testing for clinically actionable EGFR mutations in non-small cell lung cancer using the IdyllaTM platform: a real-world two-center experience in Greece.

BACKGROUND: Limited information exists on epidermal growth factor receptor (EGFR) molecular epidemiology in Greece. Next-generation sequencing (NGS) is the recommended method for EGFR genotyping in NSCLC. The Idylla Biocartis platform is a fully automated system for actionable EGFR mutation detection. RESEARCH DESIGN AND METHODS: We describe the prevalence of EGFR mutations in NSCLC patients in two high-volume clinical centers in Greece and compare key methods used for their determination. Eight hundred and fifty-seven FFPE samples from NSCLC patients were tested for EGFR mutations at University of Crete (UoC; n = 324) and at Evangelismos Hospital, Athens (Evangelismos; n = 503). RESULTS: The prevalence of EGFR mutations was 11.1% in the whole cohort (11.5% in non-squamous). The detection rate was 11.0% by NGS, 9.8% by Sanger and 11.3% by Idylla for the whole cohort (12.0% in non-squamous). The agreement between Idylla and Sanger was 93.2%. A targetable EGFR mutation was detected in 10.0% using tissue NGS alone, and in 16.0% using concurrent Idylla ctEGFR testing. CONCLUSION: The frequency of EGFR mutations was as expected for a Caucasian population. The Idylla EGFR test performance is comparable to reference methods and with a shorter TAT. Adding a concurrent plasma Idylla test to tissue NGS testing increases the detection rate of EGFR mutations in NSCLC.

Singlet oxygen-based photoelectrochemical detection of single-point mutations in the KRAS oncogene.

Single nucleotide point mutations in the KRAS oncogene occur frequently in human cancers, rendering them intriguing targets for diagnosis, early detection and personalized treatment. Current detection methods are based on polymerase chain reaction, sometimes combined with next-generation sequencing, which can be expensive, complex and have limited availability. Here, we propose a novel singlet oxygen (1O2)-based photoelectrochemical detection methodology for single-point mutations, using KRAS mutations as a case study. This detection method combines the use of a sandwich assay, magnetic beads and robust chemical photosensitizers, that need only air and light to produce 1O2, to ensure high specificity and sensitivity. We demonstrate that hybridization of the sandwich hybrid at high temperatures enables discrimination between mutated and wild-type sequences with a detection rate of up to 93.9%. Additionally, the presence of background DNA sequences derived from human cell-line DNA, not containing the mutation of interest, did not result in a signal, highlighting the specificity of the methodology. A limit of detection as low as 112 pM (1.25 ng/mL) was achieved without employing any amplification techniques. The developed 1O2-based photoelectrochemical methodology exhibits unique features, including rapidity, ease of use, and affordability, highlighting its immense potential in the field of nucleic acid-based diagnostics.

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers.

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

Performance of the cobas EZH2 mutation test on clinical samples from non-Hodgkin lymphoma patients.

OBJECTIVE: To present the technical verification and clinical validation of the companion diagnostic assay, cobas® EZH2 Mutation Test (cobas EZH2 Test), targeting gain-of-function EZH2 mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The focus is on patient clinical samples proving that the test met the performance criteria required for FDA approval of a companion diagnostic test. DESIGN: Epizyme, Inc., Eisai Co., Ltd., and Roche Molecular Systems, Inc., collaborated to develop the cobas EZH2 Test on an RT-PCR platform. The assay design needed to detect the gain-of-function EZH2 mutations found in FL and DLBCL indications. Thus, the test was optimized for investigational purposes in a clinical trial setting. Part of its technical verification included testing of patient tumor samples with a documented diagnosis of FL and DLBCL procured from commercial vendors, and the clinical validation used patient samples from the Epizyme clinical study. Both the technical performance verification method correlation study (104 clinical commercially acquired samples) and the clinical validation accuracy study (341 patient samples from the therapeutic study) used next-generation sequencing as a reference method to establish true vs. false results by cobas EZH2 Test. The reproducibility study used a 15-member panel of DNA samples with varying EZH2 mutation status from procured clinical FL and DLBCL patient samples under multiple variables. RESULTS: Single and rare, infrequent double EZH2 mutations were detected in FL and DLBCL samples. Agreements between results from cobas EZH2 and sequencing were >98% from commercial clinical samples and from the therapeutic study clinical samples. The reproducibility study obtained 178 to 180 valid results for each panel member, with an overall invalid rate of 0.37%. The agreement for each per panel member was 100%. CONCLUSION: cobas EZH2 Test data demonstrated that the test is reliable and will perform well in a commercial customer environment.

A mutation panel comprising BRAFV600E, NRASQ61R, and NRASQ61H replicated retrospective histopathological examination findings in differentiating benign goitre from malignant papillary thyroid cancer in a cohort of Malaysian patients.

Thyroid malignancy status is usually confirmed through histopathological examination (HPE) following thyroidectomy. In Malaysia, the application of molecular markers in pre-operative diagnosis of thyroid cancer remains unexplored. In this study, BRAF and NRAS gene mutation panel was assessed, and the results were compared with retrospective HPE findings. Malaysian patients with benign goitre (BTG: n=33) and papillary thyroid cancer (PTC: n=25; PTCa: n=20, PTCb: n=5) were recruited at Universiti Malaya Medical Centre from September 2019 to December 2022. PCR-direct DNA sequencing of BRAFV600, NRASG12, NRASG13, and NRASQ61 was conducted on DNA extracted from the patients' thyroid tissue specimens following thyroidectomy and HPE. BRAFV600E and NRASQ61R mutations showed absolute PTC-specificity with PTC-sensitivity of 32% and 28%, respectively. NRASQ61H demonstrated lower PTC-specificity (94%) but higher PTC-sensitivity (72%) compared to the BRAFV600E and NRASQ61R mutations. Although the NRASG12 and NRASG13 variants were absent in this study, a novel NRASV14D mutation was detected in a PTCa patient. Unlike PTCb, coexistence of BRAFV600E and NRASQ61 variants was commonly observed among the PTCa patients. Notably, all PTCb patients had NRASQ61H mutation with one patient carried both the NRASQ61H and BRAFV600E mutations. Association analysis revealed potential link between gender, BRAFV600E mutation and lymph node metastasis. In conclusion, mutation panel comprising BRAFV600E, NRASQ61R, and NRASQ61H did not discriminate the two PTC subtypes but replicated the retrospective HPE findings in differentiating BTG from PTC. The application of this mutation panel in pre-operative diagnosis of thyroid nodules requires further validation in a larger sample size, preferably incorporating fineneedle aspirate biopsies.

Mutational spectra are associated with bacterial niche.

As observed in cancers, individual mutagens and defects in DNA repair create distinctive mutational signatures that combine to form context-specific spectra within cells. We reasoned that similar processes must occur in bacterial lineages, potentially allowing decomposition analysis to detect both disruption of DNA repair processes and exposure to niche-specific mutagens. Here we reconstruct mutational spectra for 84 clades from 31 diverse bacterial species and find distinct mutational patterns. We extract signatures driven by specific DNA repair defects using hypermutator lineages, and further deconvolute the spectra into multiple signatures operating within different clades. We show that these signatures are explained by both bacterial phylogeny and replication niche. By comparing mutational spectra of clades from different environmental and biological locations, we identify niche-associated mutational signatures, and then employ these signatures to infer the predominant replication niches for several clades where this was previously obscure. Our results show that mutational spectra may be associated with sites of bacterial replication when mutagen exposures differ, and can be used in these cases to infer transmission routes for established and emergent human bacterial pathogens.

Molecular study of colorectal carcinoma and EGFR gene mutations.

The development of various human tumors can be related to the activation of the Epidermal growth factor receptor (EGFR) and its subsequent signaling pathways. There are so much alertness and awareness that has been given to the EGFR pathway recently because EGFR and some downstream components together render as targets for anticancer therapy. The EGFR pathway and its impact on colorectal carcinogenesis and assessments are the assertiveness in this paper. In this study, we took 1034 patients with colorectal carcinoma that were recorded as a medical survey we used a standard questionnaire for those patients and we used real time PCR for 30 patients from 134 cases that have colorectal carcinoma to detect if there is any mutation in the EGFR gene. We chose 4 exons for that purpose which were exons (18),(19),(20) and (21) of the EGFR gene. After deparaffinization and DNA extraction from the tissues of patients with colorectal carcinoma, we used real-time PCR technique by using (Rotor gene) kit and we were run our samples with the control group of the same patients and internal control from the kit to compare if there was any mutation but there was not any mutation in those exons of our (30) samples of paraffin-embedded (FFPE) tissues.

Comparison of Confirmed Cytology Smears and Cell Blocks for Epidermal Growth Factor Receptor Mutation Testing in Non-Small Cell Lung Cancer.

INTRODUCTION: Various cytologic specimens have been used to diagnose epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer (NSCLC). However, insufficient samples and lengthy DNA extraction procedures have led to inconsistent diagnostic results. To reduce manipulation losses and improve DNA extraction quality, we provide an improved procedure for DNA extraction from smear samples containing rare tumor cells in NSCLC. PATIENTS AND METHODS: The effectiveness of this new method for DNA extraction and diagnosis was validated in 8 patients with pleural effusion smears and formalin-fixed paraffin-embedded cell blocks, and another with 2 smears. Smear samples with <5% tumor cells were collected, and visible particles were selected for DNA extraction after centrifugation. Qiagen formalin-fixed paraffin-embedded DNA extraction kit (Qiagen) was used for DNA extraction and the procedure was modified. The EGFR mutation analysis in both types of material used the EGFR mutation analysis kit (Therascreen EGFR RGQ PCR) and real-time polymerase chain reaction (Rotor-Gene Q). RESULTS: The DNA extraction amount of the smear was 2.6 to 258.8 ng/µL, and that of the cell block was 1.4 to 139.9 ng/µL. The DNA quantity and purity of DNA extracts isolated from both sample sources were sufficient for subsequent EGFR mutation detection, where mutation rates were similar and diagnostic results were consistent when smears or cell blocks were used. CONCLUSION: This improved method demonstrates that cytology smears can be used as a test material for the detection of EGFR mutations in patients with NSCLC with sparse cells.

Diagnostic Value and Challenges of BRAF V600E Molecular Testing and Thyroid Fine-Needle Aspiration Cytology: A Retrospective Study from a Tertiary Institution in Southern Hunan Province, China.

INTRODUCTION: Combined thyroid fine-needle aspiration (FNA) cytology and valine-to-glutamate substitution at codon 600 of B-Raf proto-oncogene, serine/threonine kinase (BRAF V600E) mutation detection are procedures used for diagnosing thyroid nodules in many Chinese tertiary institutions. This retrospective study at our institution aimed to explore the effectiveness and challenges of the combined approach in diagnosing thyroid nodules and the correlation between BRAF V600E mutation status and behavior of papillary thyroid carcinoma. METHODS: Thyroid FNA cytology and BRAF V600E mutation detection results were reviewed between November 2020 and July 2022. A total of 623 patients, each of whom underwent thyroidectomy and final pathological examination after FNA cytology diagnosis, were included in the study. The relationship between the BRAF V600E mutational status and pathological parameters was analyzed using the χ2 test. The effectiveness and challenges of FNA cytology alone and the combined procedure were also evaluated based on the final pathology. RESULTS: Of 623 patients, 591 were diagnosed with papillary thyroid carcinoma (PTC), of which 456 were positive for the BRAF V600E mutation. It demonstrated near-perfect specificity for identifying PTC, and its incidence rate showed an age-specific curve with an inverted U-shaped distribution. The final pathological examination showed that the combined procedure had a higher sensitivity (83.91%) than FNA cytology alone (63.45%) for distinguishing PTC from other lesions (p < 0.001). Mutational status was associated with a larger maximum tumor diameter (p = 0.003) and a tendency of capsular invasion (p = 0.0542) but possibly unrelated to central lymph node metastasis (p = 0.1846). Nodular goiters accounted for most benign entities initially designated as Bethesda categories III-V. CONCLUSION: BRAF V600E mutational analysis complements cytopathology and improves the PTC detection rate in FNA cytology samples due to the high prevalence of the mutation in China. BRAF V600E mutation does not show a statistical correlation with tumor aggressiveness. Morphological pitfalls such as histocyte aggregation, cystic-lining cells in nodular goiters, and oncocytes in Hashimoto's thyroiditis, were overwhelmingly found in BRAF V600E-negative specimens.

Rapid assessment of 3-dimensional intra-tumor heterogeneity through cycling temperature capillary electrophoresis.

OBJECTIVE: Tumors are heterogeneous three-dimensional masses populated by numerous cell types, including distinct sub-clones of cancerous cells. Various sub-clones within the same tumor mass may respond differently to cancer treatment, and intra-tumor heterogeneity contributes to acquired therapeutic resistance. Thus, one tissue biopsy will in most cases not be representative of the entire genetic landscape of a tumor mass. In this study, we aimed to establish an easily accessible, low cost method to address intra-tumor heterogeneity in three dimensions, for a limited number of DNA alterations. RESULTS: This study includes analyses of the three-dimensional (3D) distribution of DNA mutations in human colon cancer and mouse mammary gland tumor tissue samples. We used laser capture microdissection for the unbiased collection of tissue in several XY-planes throughout the tumor masses. Cycling temperature capillary electrophoresis was used to determine mutant allele frequency. High-resolution distribution maps of KRAS and Trp53 mutations were generated for each XY-plane in human and mouse tumor samples, respectively. To provide a holistic interpretation of the mutation distribution, we generated interactive 3D heatmaps giving an easily interpretable understanding of the spatial distribution of the analyzed mutations. The method described herein provides an accessible way of describing intra-tumor heterogeneity for a limited number of mutations.

Reliability of panel-based mutational signatures for immune-checkpoint-inhibition efficacy prediction in non-small cell lung cancer.

OBJECTIVES: Mutational signatures (MS) are gaining traction for deriving therapeutic insights for immune checkpoint inhibition (ICI). We asked if MS attributions from comprehensive targeted sequencing assays are reliable enough for predicting ICI efficacy in non-small cell lung cancer (NSCLC). METHODS: Somatic mutations of m = 126 patients were assayed using panel-based sequencing of 523 cancer-related genes. In silico simulations of MS attributions for various panels were performed on a separate dataset of m = 101 whole genome sequenced patients. Non-synonymous mutations were deconvoluted using COSMIC v3.3 signatures and used to test a previously published machine learning classifier. RESULTS: The ICI efficacy predictor performed poorly with an accuracy of 0.51-0.09+0.09, average precision of 0.52-0.11+0.11, and an area under the receiver operating characteristic curve of 0.50-0.09+0.10. Theoretical arguments, experimental data, and in silico simulations pointed to false negative rates (FNR) related to panel size. A secondary effect was observed, where deconvolution of small ensembles of point mutations lead to reconstruction errors and misattributions. CONCLUSION: MS attributions from current targeted panel sequencing are not reliable enough to predict ICI efficacy. We suggest that, for downstream classification tasks in NSCLC, signature attributions be based on whole exome or genome sequencing instead.

Metanephric stromal tumor in an adult with PDGFRA mutation: a case report.

BACKGROUND: Metanephric stromal tumors (MST) are rare benign renal tumors that mainly occur in infants and children. Approximately 72% of MST in children have the B-Raf proto-oncogene serine/threonine kinase (BRAF) V600E mutation. To date, only five cases of adult MSTs have been reported and no clear genetic alterations have been found. CASE PRESENTATION: We report a case of MST in a 45-year-old woman who complained of left lower back pain for a week, accompanied by hypertension (150/79 mmHg). Magnetic resonance imaging (MRI) showed an abnormally enhanced nodule (1.1 cm in the middle of the left kidney), which was histopathologically consistent with an MST. The BRAF V600E mutation was not detected in tumor cells using PCR and next-generation sequencing (NGS). However, a platelet-derived growth factor receptor alpha (PDGFRA) mutation was detected in this case using NGS. The patient showed no recurrence or metastasis nine months after partial nephrectomy, and her blood pressure was consistently normal. CONCLUSION: This is the first report of alterations in PDGFRA in MSTs. This result advances our knowledge of genetic variations in adult MSTs, which may have different gene alterations from MSTs in children.

Significance of FNAC, BRAF mutation, and intraoperative frozen section in surgical decision-making of thyroid nodules.

BACKGROUND: A preoperative method is desired to discriminate benign from malignant thyroid nodules. This retrospective study evaluated the diagnostic performance of BRAF (B-Raf proto-oncogene) mutation (BRAFV600E ) positivity and fine-needle aspiration cytology (FNAC) relative to intraoperative frozen section pathology. METHODS: Patients underwent preoperative FNAC of thyroid nodules. Cytology specimens were classified according to the Bethesda System for Reporting Thyroid Cytopathology (BSRTC), and analyzed for BRAFV600E using an amplification-refractory mutation system (ARMS). Thyroid tissue was surgically removed and frozen sections processed for histology. The sensitivities and specificities of each analysis were compared, alone and in combination. RESULTS: Among 346 patients, 333/358 FNACs (93%) showed malignant nodules; 322 (93%) patients received a pathological diagnosis of papillary thyroid carcinoma (PTC). The sensitivity and specificity of BSRTC VI for malignancy was the highest among the BSRTC categories. Compared with FNAC, the BRAFV600E analysis had significantly higher sensitivity and specificity. The diagnostic efficacy of frozen section pathology was significantly higher than that of either BSRTC category or BRAFV600E analysis alone. Combining methods variably improved diagnostic performance. BRAFV600E was not associated with capsule infiltration, neurovascular infiltration, mono- or multifocal PTC, lymph node metastasis, or clinical stage. CONCLUSION: The diagnostic performance of preoperative BRAFV600E determination was better than that of the BSRTC-FNAC category; combining both improved sensitivity and specificity. Patients with positive malignancy scores from both should be recommended for surgery; those with negative scores require close monitoring. Surgical treatment should include comprehensive intraoperative frozen section assessment. BRAF mutations cannot indicate aggressive treatment.

FACILITATE: A real-world, multicenter, prospective study investigating the utility of a rapid, fully automated real-time PCR assay versus local reference methods for detecting epidermal growth factor receptor variants in NSCLC.

Accurate testing for epidermal growth factor receptor (EGFR) variants is essential for informing treatment decisions in non-small cell lung cancer (NSCLC). Automated diagnostic workflows may allow more streamlined initiation of targeted treatments, where appropriate, while comprehensive variant analysis is ongoing. FACILITATE, a real-world, prospective, multicenter, European study, evaluated performance and analytical turnaround time of the Idylla™ EGFR Mutation Test compared with local reference methods. Sixteen sites obtained formalin-fixed paraffin-embedded biopsy samples with ≥ 10% neoplastic cells from patients with NSCLC. Consecutive 5 µm sections from patient samples were tested for clinically relevant NSCLC-associated EGFR variants using the Idylla™ EGFR Mutation Test and local reference methods; performance (concordance) and analytical turnaround time were compared. Between January 2019 and November 2020, 1,474 parallel analyses were conducted. Overall percentage agreement was 97.7% [n = 1,418; 95% confidence interval (CI): 96.8-98.3], positive agreement, 87.4% (n = 182; 95% CI: 81.8-91.4) and negative agreement, 99.2% (n = 1,236; 95% CI: 98.5-99.6). There were 38 (2.6%) discordant cases. Ninety percent of results were returned with an analytical turnaround time of within 1 week using the Idylla™ EGFR Mutation Test versus ∼22 days using reference methods. The Idylla™ EGFR Mutation Test performed well versus local methods and had shorter analytical turnaround time. The Idylla™ EGFR Mutation Test can thus support application of personalized medicine in NSCLC.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Annotation-Free Deep Learning-Based Prediction of Thyroid Molecular Cancer Biomarker BRAF (V600E) from Cytological Slides.

Thyroid cancer is the most common endocrine cancer. Papillary thyroid cancer (PTC) is the most prevalent form of malignancy among all thyroid cancers arising from follicular cells. Fine needle aspiration cytology (FNAC) is a non-invasive method regarded as the most cost-effective and accurate diagnostic method of choice in diagnosing PTC. Identification of BRAF (V600E) mutation in thyroid neoplasia may be beneficial because it is specific for malignancy, implies a worse prognosis, and is the target for selective BRAF inhibitors. To the authors' best knowledge, this is the first automated precision oncology framework effectively predict BRAF (V600E) immunostaining result in thyroidectomy specimen directly from Papanicolaou-stained thyroid fine-needle aspiration cytology and ThinPrep cytological slides, which is helpful for novel targeted therapies and prognosis prediction. The proposed deep learning (DL) framework is evaluated on a dataset of 118 whole slide images. The results show that the proposed DL-based technique achieves an accuracy of 87%, a precision of 94%, a sensitivity of 91%, a specificity of 71% and a mean of sensitivity and specificity at 81% and outperformed three state-of-the-art deep learning approaches. This study demonstrates the feasibility of DL-based prediction of critical molecular features in cytological slides, which not only aid in accurate diagnosis but also provide useful information in guiding clinical decision-making in patients with thyroid cancer. With the accumulation of data and the continuous advancement of technology, the performance of DL systems is expected to be improved in the near future. Therefore, we expect that DL can provide a cost-effective and time-effective alternative tool for patients in the era of precision oncology.

Retinoblastoma: From genes to patient care.

Retinoblastoma is the most common paediatric neoplasm of the retina, and one of the earliest model of cancer genetics since the identification of the master tumour suppressor gene RB1. Tumorigenesis has been shown to be driven by pathogenic variants of the RB1 locus, but also genomic and epigenomic alterations outside the locus. The increasing knowledge on this "mutational landscape" is used in current practice for precise genetic testing and counselling. Novel methods provide access to pre-therapeutic tumour DNA, by isolating cell-free DNA from aqueous humour or plasma. This is expected to facilitate assessment of the constitutional status of RB1, to provide an early risk stratification using molecular prognostic markers, to follow the response to the treatment in longitudinal studies, and to predict the response to targeted therapies. The aim of this review is to show how molecular genetics of retinoblastoma drives diagnosis, treatment, monitoring of the disease and surveillance of the patients and relatives. We first recap the current knowledge on retinoblastoma genetics and its use in every-day practice. We then focus on retinoblastoma subgrouping at the era of molecular biology, and the expected input of cell-free DNA in the field.