An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.

Guidance Statement On BRCA1/2 Tumor Testing in Ovarian Cancer Patients.

The approval, in 2015, of the first poly (adenosine diphosphate-ribose) polymerase inhibitor (PARPi; olaparib, Lynparza) for platinum-sensitive relapsed high-grade ovarian cancer with either germline or somatic BRCA1/2 deleterious mutations is changing the way that BRCA1/2 testing services are offered to patients with ovarian cancer. Ovarian cancer patients are now being referred for BRCA1/2 genetic testing for treatment decisions, in addition to familial risk estimation, and irrespective of a family history of breast or ovarian cancer. Furthermore, testing of tumor samples to identify the estimated 3%-9% of patients with somatic BRCA1/2 mutations who, in addition to germline carriers, could benefit from PARPi therapy is also now being considered. This new testing paradigm poses some challenges, in particular the technical and analytical difficulties of analyzing chemically challenged DNA derived from formalin-fixed, paraffin-embedded specimens. The current manuscript reviews some of these challenges and technical recommendations to consider when undertaking BRCA1/2 testing in tumor tissue samples to detect both germline and somatic BRCA1/2 mutations. Also provided are considerations for incorporating genetic analysis of ovarian tumor samples into the patient pathway and ethical requirements.

A comparative study of germline BRCA1 and BRCA2 mutation screening methods in use in 20 European clinical diagnostic laboratories.

BACKGROUND: Thousands of clinically relevant variations in BRCA1 and BRCA2 have been discovered and this poses a significant challenge with respect to the accurate detection, analysis turn-around time, characterisation and interpretation of these sequence variants. METHODS: We evaluated the performance of different BRCA1/2 gene testing practices in routine diagnostic use in 20 European laboratories, with a focus on next-generation sequencing-based strategies as this is the technical approach implemented by or under adoption by most European clinical laboratories. Participant laboratories, selected on expertise and diagnostic service quality, tested 10 identical DNA samples containing a range of challenging pathogenic variants. RESULTS: A small number of errors in the detection of pathogenic and significant variants were identified (2.6% diagnostic error rate). There was a high degree of concordance (>97%) across all laboratories for all variants detected. No systematic technical flaw was identified in the strategies employed across the participating laboratories. CONCLUSIONS: The discrepancies identified are most likely due to human error or the way the methodology has been implemented locally, for example, next-generation sequencing bioinformatics pipelines, rather than technical limitations of the methods. The choice of BRCA1/2 testing method will therefore depend on multiple factors including required throughput and turn-around times, access to equipment, expertise and budget.

The First Nationwide Multicenter Prevalence Study of Germline BRCA1 and BRCA2 Mutations in Chinese Ovarian Cancer Patients.

OBJECTIVE: Subjects with germline BRCA1/2 mutations (gBRCAm) have an increased risk of developing ovarian cancer and enhanced sensitivity to platinum-containing agents and PARP (poly[ADP-ribose] polymerase) inhibitors. BRCA mutations in Asian patients are poorly understood compared with other populations. We aimed to investigate gBRCAm prevalence and characteristics in Chinese ovarian cancer patients. METHODS: We conducted the first nationwide multicenter gBRCAm prevalence study in China. Eight hundred twenty-six unselected ovarian cancer patients from 5 clinical centers were enrolled and tested for gBRCAm status. Medical data including age, family history, previous treatments, clinical diagnosis, histopathologic diagnosis, tumor grade, platinum sensitivity, and CA-125 test result were reviewed and collected. RESULTS: Prevalence rate or gBRCAm was determined as 28.5%, with 20.8% of patients harboring BRCA1 mutation and 7.6% harboring BRCA2 mutation. The group had a higher percentage of high-grade serous (73.0%), late-stage (III and IV [85.5%]) patients and a younger median age at diagnosis (52 years) compared with other reported studies. Twnety-seven BRCA1 and 17 BRCA2 mutations have not been reported previously in public databases or the literature. Statistically significant correlations were observed between gBRCAm status and family history (P < 0.001), gBRCAm status, and tumor stage (P = 0.02). A numerical higher prevalence of gBRCAm in patients with high-grade serous histopathology (30.9%), platinum-sensitive phenotype (34%), and late-line chemotherapy was observed. CONCLUSIONS: Germline BRCA1/2 mutations is common in Chinese ovarian cancer patients. This study implies that all ovarian patients should be tested for gBRCAm status regardless of family history and histopathology.

Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples.

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy.

A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.

BACKGROUND: Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue. METHODS: We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated. RESULTS: Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples. CONCLUSIONS: Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients.

Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum.

The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.

AIMS: To evaluate the suitability of malignant pleural effusion (MPE) and plasma as surrogate samples for epidermal growth factor receptor (EGFR) mutation detection, and compare three different detection methods. METHODS: Matched tissue and plasma samples were collected from patients with advanced non-small cell lung cancer (NSCLC) (stage IIIB/IV adenocarcinoma/adenosquamous carcinoma), with matched MPE samples collected from a subgroup. DNA was extracted from tissue, MPE cell block, MPE supernatant and plasma before mutation detection by amplification refractory mutation system (ARMS) (all samples), Sanger sequencing and mutant-specific immunohistochemistry (IHC) (tissue and MPE cell blocks only). RESULTS: Sensitivity of MPE cell block, MPE supernatant and plasma versus tissue: 81.8% (9/11), 63.6% (7/11) and 67.5% (27/40); specificity was 80.0% (8/10), 100% (10/10) and 100% (46/46), respectively. Sensitivity of Sanger sequencing versus ARMS: 81.8% (27/33) for tissue, 40% (4/10) for MPE cell blocks; specificity was 100% (36/36 and 12/12) for both. Sensitivity of mutant-specific IHC versus ARMS: 54.8% (17/31) for tissue, 50.0% (6/12) for MPE cell blocks; specificity was 97.1% (34/35) and 100% (14/14), respectively. CONCLUSIONS: MPE and plasma are valid surrogates for NSCLC tumour EGFR mutation detection when tissue is not available. ARMS is most suitable for mutation detection in tissue and MPE cell blocks; however, mutant-specific IHC could be a complementary method when DNA-based molecular testing is unavailable.

EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples.

AIMS: Activating mutations in the gene encoding epidermal growth factor receptor (EGFR) can confer sensitivity to EGFR tyrosine kinase inhibitors such as gefitinib in patients with advanced non-small-cell lung cancer. Testing for mutations in EGFR is therefore an important step in the treatment-decision pathway. We reviewed reported methods for EGFR mutation testing in patients with lung cancer, initially focusing on studies involving standard tumour tissue samples. We also evaluated data on the use of cytology samples in order to determine their suitability for EGFR mutation analysis. METHODS: We searched the MEDLINE database for studies reporting on EGFR mutation testing methods in patients with lung cancer. RESULTS: Various methods have been investigated as potential alternatives to the historical standard for EGFR mutation testing, direct DNA sequencing. Many of these are targeted methods that specifically detect the most common EGFR mutations. The development of targeted mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples. The use of screening methods, subsequent to sample micro dissection, has also ensured that identification of more rare, uncommon mutations is now feasible. Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed. CONCLUSIONS: Several different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations. Evidence published to date suggests cytology samples are viable alternatives for mutation testing when tumour tissue samples are not available.

A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples.

BACKGROUND: We have compared mutation analysis by DNA sequencing and Amplification Refractory Mutation System™ (ARMS™) for their ability to detect mutations in clinical biopsy specimens. METHODS: We have evaluated five real-time ARMS assays: BRAF 1799T>A, [this includes V600E and V600K] and NRAS 182A>G [Q61R] and 181C>A [Q61K] in melanoma, EGFR 2573T>G [L858R], 2235-2249del15 [E746-A750del] in non-small-cell lung cancer, and compared the results to DNA sequencing of the mutation 'hot-spots' in these genes in formalin-fixed paraffin-embedded tumour (FF-PET) DNA. RESULTS: The ARMS assays maximised the number of samples that could be analysed when both the quality and quantity of DNA was low, and improved both the sensitivity and speed of analysis compared with sequencing. ARMS was more robust with fewer reaction failures compared with sequencing and was more sensitive as it was able to detect functional mutations that were not detected by DNA sequencing. DNA sequencing was able to detect a small number of lower frequency recurrent mutations across the exons screened that were not interrogated using the specific ARMS assays in these studies. CONCLUSIONS: ARMS was more sensitive and robust at detecting defined somatic mutations than DNA sequencing on clinical samples where the predominant sample type was FF-PET.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244).

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Epigenetic silencing of the endothelin-B receptor gene in non-small cell lung cancer.

Endothelin-1 is overexpressed in several tumor types. Activation of the endothelin-A (ETA) receptor may promote cell growth, angiogenesis and invasion, and inhibits the apoptotic process, while activation of the endothelin-B (ETB) receptor may induce cell death by apoptosis and inhibit tumor progression. Hypermethylation and subsequent silencing of the ETB receptor gene promoter has been reported in some cancer types. As the endothelin pathway is subject to research for pharmacological cancer treatment, we investigated the extent of epigenetic deregulation of the ETB receptor gene in non-small cell lung cancer (NSCLC). We scanned 64 NSCLC paired tumor/normal surgical specimens for the ETB receptor promoter for methylation by developing four pyrosequencing assays that covered 24 CpGs. The ETB receptor promoter was significantly hypermethylated in 31 (48%) of tumor samples, presenting considerably higher methylation in 22/24 CpG sites compared with the normal counterpart tissues. ETB receptor mRNA levels were reduced in all lung tumors compared with normal adjacent lung tissue, indicating the potentially important involvement of this gene in lung cancer development. Furthermore, tumor samples with ETB receptor gene methylation tended to have lower receptor mRNA levels compared with unmethylated tumor specimens, suggesting a primary epigenetic role in ETB receptor silencing. Our results point to a significant involvement of ETB receptor epigenetic deregulation in the pathogenesis of lung cancer making the gene a promising candidate biomarker for response to regimens modulating the endothelin axis.

Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer.

PURPOSE: The phase III Iressa Survival Evaluation in Lung Cancer (ISEL) trial compared gefitinib with placebo in 1,692 patients with refractory advanced non-small-cell lung cancer. We analyzed ISEL tumor biopsy samples to examine relationships between biomarkers and clinical outcome after gefitinib treatment in a placebo-controlled setting. METHODS: Biomarkers included epidermal growth factor receptor (EGFR) gene copy number by fluorescence in situ hybridization (n = 370); EGFR (n = 379) and phosphorylated Akt (p-Akt) protein expression (n = 382) by immunohistochemistry; and mutations in EGFR (n = 215), KRAS (n = 152), and BRAF (n = 118). RESULTS: High EGFR gene copy number was a predictor of a gefitinib-related effect on survival (hazard ratio [HR], 0.61 for high copy number and HR, 1.16 for low copy number; comparison of high v low copy number HR, P = .045). EGFR protein expression was also related to clinical outcome (HR for positive, 0.77; HR for negative, 1.57; comparison of high v low protein expression HR, P = .049). Patients with EGFR mutations had higher response rates than patients without EGFR mutations (37.5% v 2.6%); there were insufficient data for survival analysis. No relationship was observed between p-Akt protein expression and survival outcome, and the limited amount of data collected for KRAS and BRAF mutations prevented any meaningful evaluation of clinical outcomes in relation to these mutations. CONCLUSION: EGFR gene copy number was a predictor of clinical benefit from gefitinib in ISEL. Additional studies are warranted to assess these biomarkers fully for the identification of patients most likely to benefit from gefitinib treatment.