Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma.

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Human papillomavirus (HPV) DNA methylation changes in HPV-associated head and neck cancer.

Despite the rising incidence, currently, there are no early detection methods for HPV-driven HNC (HPV-HNC). Cervical cancer studies suggest that HPV DNA methylation changes can be used as a biomarker to discriminate cancer patients from HPV-infected individuals. As such, this study was designed to establish a protocol to evaluate DNA methylation changes in HPV late genes and long control region (LCR) in saliva samples of HPV-HNC patients and HPV-positive controls. Higher methylation levels were detected in HPV late genes (L1 and L2) in both tumour and saliva samples of HPV-HNC patients compared with HPV-positive controls. Moreover, methylation patterns between tumours and corresponding saliva samples were observed to have a strong correlation (Passing-Bablok regression analysis; τ = 0.7483, P < 0.0001). Considering the differences between HNC and controls in methylation levels in late genes, and considering primer amplification efficiencies, 13 CpG sites located at L1 and L2 genes were selected for further evaluation. A total of 18 HNC saliva samples and 10 control saliva samples were assessed for the methylation levels in the selected sites. From the CpG sites evaluated statistically significant differences were identified for CpG sites at L2-CpG 6 (P = 0.0004), L1-CpG 3 (P = 0.0144), L1-CpG 2 (P = 0.0395) and L2-CpG 19 (P = 0.0455). Our pilot data indicate that higher levels of DNA methylation in HPV late genes are indicative of HPV-HNC risk, and it is a potential supplementary biomarker for salivary HPV detection-based HPV-HNC screening.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

Prognostic value of integrating circulating tumour cells and cell-free DNA in non-small cell lung cancer.

Background: Non-small cell lung cancer (NSCLC) often presents at an incurable stage, and majority of patients will be considered for palliative treatment at some point in their disease. Despite recent advances, the prognosis remains poor, with a median overall survival of 12-18 months. Liquid biopsy-based biomarkers have emerged as potential candidates for predicting prognosis and response to therapy in NSCLC patients. This pilot study evaluated whether combining circulating tumour cells and clusters (CTCs) and cell-free DNA (cfDNA) can predict progression-free survival (PFS) in NSCLC patients. Methods: CTC and cfDNA/ctDNA from advanced stage NSCLC patients were measured at study entry (T0) and 3-months post-treatment (T1). CTCs were enriched using a spiral microfluidic chip and characterised by immunofluorescence. ctDNA was assessed using an UltraSEEK® Lung Panel. Kaplan-Meier plots were generated to investigate the contribution of the presence of CTC/CTC clusters and cfDNA for PFS. Cox proportional hazards analysis compared time to progression versus CTC/CTC cluster counts and cfDNA levels. Results: Single CTCs were found in 14 out of 25 patients, while CTC clusters were found in 8 out of the 25 patients at T0. At T1, CTCs were found in 7 out of 18 patients, and CTC clusters in 1 out of the 18 patients. At T0, CTC presence and the combination of CTC cluster counts with cfDNA levels were associated with shorter PFS, p = 0.0261, p = 0.0022, respectively. Conclusions: Combining CTC cluster counts and cfDNA levels could improve PFS assessment in NSCLC patients. Our results encourage further investigation on the combined effect of CTC/cfDNA as a prognostic biomarker in a large cohort of advanced stage NSCLC patients.

Genomic profiling using the UltraSEEK panel identifies discordancy between paired primary and breast cancer brain metastases and an association with brain metastasis-free survival.

PURPOSE: Brain metastases (BM) are an increasing clinical problem. This study aimed to assess paired primary breast cancers (BC) and BM for aberrations within TP53, PIK3CA, ESR1, ERBB2 and AKT utilising the MassARRAY® UltraSEEK® technology (Agena Bioscience, San Diego, USA). METHODS: DNA isolated from 32 paired primary BCs and BMs was screened using the custom UltraSEEK® Breast Cancer Panel. Data acquisition and analysis was performed by the Agena Bioscience Typer software v4.0.26.74. RESULTS: Mutations were identified in 91% primary BCs and 88% BM cases. TP53, AKT1, ESR1, PIK3CA and ERBB2 genes were mutated in 68.8%, 37.5%, 31.3%, 28.1% and 3.1% respectively of primary BCs and in 59.4%, 37.5%, 28.1%, 28.1% and 3.1% respectively of BMs. Differences in the mutations within the 5 genes between BC and paired BM were identified in 62.5% of paired cases. In primary BCs, ER-positive/HER2-negative cases harboured the most mutations (70%), followed by ER-positive/HER2-positive (15%) and triple-negatives (13.4%), whereas in BMs, the highest number of mutations was observed in triple-negative (52.5%), followed by ER-positive/HER2-negative (35.6%) and ER-negative/HER2-positive (12%). There was a significant association between the number of mutations in the primary BC and breast-to-brain metastasis-free survival (p = 0.0001) but not with overall survival (p = 0.056). CONCLUSION: These data demonstrate the discordancy between primary BC and BM, as well as the presence of clinically important, actionable mutations in BCBM. The UltraSEEK® Breast Cancer Panel provides a tool for BCBM that can be utilised to direct more tailored treatment decisions and for clinical studies investigating targeted agents.

The identification of circulating tumour DNA using MassARRAY technology in non-small-cell lung cancer (NSCLC).

OBJECTIVES: Attaining tumour material from lung cancer patients can be challenging with limited sample availability. Therefore, non-invasive means of assessing tumour material is becoming increasingly more important. Circulating tumour DNA (ctDNA), extracted from a blood sample is appealing for the patient, and can be performed serially over the course of treatment. MATERIALS AND METHODS: Here, we describe an approach for profiling the blood samples of 103 NSCLC patients for 73 variants in ctDNA across a panel of actionable lung cancer mutations using the UltraSEEK lung Panel (Agena Biosciences). RESULTS: Our cross-sectional study showed tumour and blood concordance in the detection of KRAS mutations (G12C, G12D, G12A/V, G12R, G12RC, Q61H) in 17/27 (63%), EGFR mutations (e746_a750del, e747_A750, T790M, L861Q) in 16/20 (80%) with additional PIK3CA_p545K mutations across both cohorts. In patients without reported tumour mutations, 11/56 (19.6%) presented with plasma mutations across EGFR, KRAS and PIK3CA. Where ctDNA mutations were measured longitudinally (n = 4 patients), the individual mutations mirrored the response to therapy/progression of disease. CONCLUSION: Whilst preliminary, this study demonstrates the utility of detecting clinically actionable mutations in the blood samples of NSCLC patients at the time of presentation, and over the course of therapy.

Discovery of Targetable Genetic Alterations in NSCLC Patients with Different Metastatic Patterns Using a MassARRAY-Based Circulating Tumor DNA Assay.

Circulating tumor DNA (ctDNA) has shown great promise as a minimally invasive liquid biopsy for personalized cancer diagnostics especially among metastatic patients. Here, we used a novel sensitive assay to detect clinically relevant mutations in ctDNA in blood plasma from metastatic non-small cell lung cancer (NSCLC) patients, including patients with a limited oligo-brain metastatic disease. We analyzed 66 plasma samples from 56 metastatic NSCLC patients for 74 hotspot mutations in five genes commonly mutated in NSCLC using a novel MassARRAY-based lung cancer panel with a turnaround time of only 3 days. Mutations in plasma DNA could be detected in 28 out of 56 patients (50.0%), with a variant allele frequency (VAF) ranging between 0.1% and 5.0%. Mutations were detected in 50.0% of patients with oligo-brain metastatic disease, although the median VAF was lower (0.4%) compared to multi-brain metastatic patients (0.9%) and patients with extra-cranial metastatic progression (1.2%). We observed an overall concordance of 86.4% (n = 38/44) for EGFR status between plasma and tissue. The MassARRAY technology can detect clinically relevant mutations in plasma DNA from metastatic NSCLC patients including patients with limited, oligo-brain metastatic disease.

Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer.

BACKGROUNDCurrent methods for the detection and surveillance of bladder cancer (BCa) are often invasive and/or possess suboptimal sensitivity and specificity, especially in early-stage, minimal, and residual tumors.METHODSWe developed an efficient method, termed utMeMA, for the detection of urine tumor DNA methylation at multiple genomic regions by MassARRAY. We identified the BCa-specific methylation markers by combined analyses of cohorts from Sun Yat-sen Memorial Hospital (SYSMH), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) database. The BCa diagnostic model was built in a retrospective cohort (n = 313) and validated in a multicenter, prospective cohort (n = 175). The performance of this diagnostic assay was analyzed and compared with urine cytology and FISH.RESULTSWe first discovered 26 significant methylation markers of BCa in combined analyses. We built and validated a 2-marker-based diagnostic model that discriminated among patients with BCa with high accuracy (86.7%), sensitivity (90.0%), and specificity (83.1%). Furthermore, the utMeMA-based assay achieved a great improvement in sensitivity over urine cytology and FISH, especially in the detection of early-stage (stage Ta and low-grade tumor, 64.5% vs. 11.8%, 15.8%), minimal (81.0% vs. 14.8%, 37.9%), residual (93.3% vs. 27.3%, 64.3%), and recurrent (89.5% vs. 31.4%, 52.8%) tumors. The urine diagnostic score from this assay was better associated with tumor malignancy and burden.CONCLUSIONUrine tumor DNA methylation assessment for early diagnosis, minimal, residual tumor detection and surveillance in BCa is a rapid, high-throughput, noninvasive, and promising approach, which may reduce the burden of cystoscopy and blind second surgery.FUNDINGThis study was supported by the National Key Research and Development Program of China and the National Natural Science Foundation of China.

Pre-analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients.

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next-generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre-analytical variable.

Genomic Analysis of Circulating Tumor DNA Using a Melanoma-Specific UltraSEEK Oncogene Panel.

The analysis of circulating tumor DNA provides a minimally invasive molecular interrogation that has the potential to guide treatment selection and disease monitoring. Here, the authors evaluated a custom UltraSEEK melanoma panel for the MassARRAY system, probing for 61 mutations over 13 genes. The analytical sensitivity and clinical accuracy of the UltraSEEK melanoma panel was compared with droplet digital PCR. The blinded analysis of 68 mutations detected in 48 plasma samples from stage IV melanoma patients revealed a concordance of 88% between the two platforms. Further comparison of both methods for the detection of BRAF V600E mutations in 77 plasma samples demonstrated a Cohen's κ of 0.826 (bias-corrected and accelerated 95% CI, 0.669-0.946). These results indicate that the UltraSEEK melanoma panel is as sensitive as droplet digital PCR for the detection of circulating tumor DNA in this cohort of patients but highlight the need for detected variants to be confirmed orthogonally to mitigate any false-positive results. The MassARRAY system enables rapid and sensitive genotyping for the detection of multiple melanoma-associated mutations in plasma.

Assessment of common somatic mutations of EGFR, KRAS, BRAF, NRAS in pulmonary non-small cell carcinoma using iPLEX® HS, a new highly sensitive assay for the MassARRAY® System.

Increased early detection and personalized therapy for lung cancer have coincided with greater use of minimally invasive sampling techniques such as endobronchial ultrasound-guided biopsy (EBUS), endoscopic ultrasound-guided biopsy (EUS), and navigational biopsy, as well as thin needle core biopsies. As many lung cancer patients have late stage disease and other comorbidities that make open surgical procedures hazardous, the least invasive biopsy technique with the highest potential specimen yield is now the preferred first diagnostic study. However, use of these less invasive procedures generates significant analytical challenges for the laboratory, such as a requirement for robust detection of low level somatic mutations, particularly when the starting sample is very small or demonstrates few intact tumor cells. In this study, we assessed 179 clinical cases of non-small cell lung carcinoma (NSCLC) that had been previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, iPLEX® HS panel for the MassARRAY® System (Agena Bioscience, San Diego, CA). This highly sensitive system identified approximately 10% more KRAS, NRAS, EGFR and BRAF mutations than were detected by the original test platform, which had a sensitivity range of 5-10% variant allele frequency (VAF).

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer.

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86-96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof-of-principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.

Genotyping for Glycophorin GYP(B-A-B) Hybrid Genes Using a Single Nucleotide Polymorphism-Based Algorithm by Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry.

The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3' breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

Molecular typing for the Indian blood group associated 252G>C single nucleotide polymorphism in a selected cohort of Australian blood donors.

BACKGROUND: The Indian blood group antigens, In(a) and In(b), are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The In(b) antigen is a high frequency antigen present in all populations, while the frequency of the antithetical In(a) ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the IN*A and IN*B alleles in a blood donor cohort. MATERIALS AND METHODS: Donor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques. RESULTS: In this study group, 149 IN*B homozygous and 2 IN*A/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate IN*A and IN*B alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both In(a) and In(b) antigens were present as predicted. DISCUSSION: Genotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the IN*A and IN*B alleles were discriminated on the basis of their melting properties. The In(a) frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance.

Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. CONCLUSIONS/SIGNIFICANCE: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor.

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy.

A high-throughput panel for identifying clinically relevant mutation profiles in melanoma.

Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs.

Sensitive detection of BCR-ABL1 mutations in patients with chronic myeloid leukemia after imatinib resistance is predictive of outcome during subsequent therapy.

PURPOSE: BCR-ABL1 mutation analysis is recommended to facilitate selection of appropriate therapy for patients with chronic myeloid leukemia after treatment with imatinib has failed, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. However, mutations could be present below the detection limit of conventional direct sequencing. We developed a sensitive, multiplexed mass spectrometry assay (detection limit, 0.05% to 0.5%) to determine the impact of low-level mutations after imatinib treatment has failed. PATIENTS AND METHODS: Mutation status was assessed in 220 patients treated with nilotinib or dasatinib after they experienced resistance to imatinib. RESULTS: Mutations were detected by sequencing in 128 patients before commencing nilotinib or dasatinib therapy (switchover). In 64 patients, 132 additional low-level mutations were detected by mass spectrometry alone (50 of 132 mutations were resistant to nilotinib and/or dasatinib). When patients received the inhibitor for which the mutation confers resistance, 84% of the low-level resistant mutations rapidly became dominant clones detectable by sequencing, including 11 of 12 T315I mutations. Subsequent complete cytogenetic response rates were lower for patients with resistant mutations at switchover detected by sequencing (0%) or mass spectrometry alone (16%) compared with patients with other mutations or no mutations (41% and 49%, respectively; P < .001). Failure-free survival among the 100 patients with chronic phase chronic myeloid leukemia when resistant mutations were detected at switchover by sequencing or mass spectrometry alone was 0% and 0% compared with 51% and 45% for patients with other mutations or no mutations (P = .003). CONCLUSION: Detection of low-level mutations after imatinib resistance offers critical information to guide subsequent therapy selection. If an inappropriate kinase inhibitor is selected, there is a high risk of treatment failure with clonal expansion of the resistant mutant.

Evaluation of non-invasive prenatal RHD genotyping of the fetus.

OBJECTIVE: To evaluate a non-invasive molecular test using free circulating fetal DNA in maternal plasma to predict the fetal RHD type. DESIGN: A prospective cohort study. PARTICIPANTS AND SETTING: Venous blood samples were collected from 140 Rhesus (Rh) D-negative women booked for antenatal care in two tertiary maternity hospitals in Sydney and Brisbane between November 2006 and April 2008. Cell-free DNA, including free maternal and fetal DNA, was extracted from maternal plasma in the tertiary Australian Red Cross Blood Service laboratory, and three exon regions of the RHD gene were amplified. MAIN OUTCOME MEASURES: Comparison of the predicted fetal RHD status and the infant's RhD serotype. Secondary analysis involved using SRY and RASSF1A assays as internal controls to confirm the presence of fetal DNA in RHD-negative samples. RESULTS: Of 140 samples tested, results for RHD status were assigned for 135, and all 135 predictions were correct. A result was not assigned in five cases: three did not meet strict threshold criteria for classification, and two were due to RHD variants. Fetal SRY status was correctly predicted in 137 of 140 cases. In 16 samples typed both RHD- and SRY-negative, a positive RASSF1A result verified the presence of fetal DNA. CONCLUSIONS: Non-invasive testing of multiple exons provides a robust method of assessing fetal RHD status, and provides a safer alternative to amniocentesis for the management of RhD-negative pregnant women who are isoimmunised.

Identification of candidate tumor suppressor genes inactivated by promoter methylation in melanoma.

Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation. To identify novel methylated TSGs in melanoma, we carried out global mRNA expression profiling on a panel of 12 melanoma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Trichostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays. After qRT-PCR confirmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C, RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-specific cleaved amplification products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed reduced mRNA expression in 35-59% of the melanoma cell lines compared to melanocytes and which was correlated with a high proportion of promoter methylation (>40-60%). The same genes also showed extensive promoter methylation in 6-25% of the tumor samples, thus confirming them as novel candidate TSGs in melanoma.