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The standard of care for women presenting with early stage- ER-positive breast cancer (BC) following "curative" surgery has been 5 years of TAM. Adjuvant treatment with TAM has changed the natural history of BC, producing a significant reduction in 5- and 10-year recurrence rates; however, because of its adverse effects, many women (approx. 40%) do not complete the recommended 5 years of treatment. Furthermore, since TAM is a pro-drug that needs to be converted to endoxifen to be effective, inter-individual variability in endogenous enzymatic activity (i.e., CYP2D6) can affect endoxifen exposure. Certain drugs (e.g., SSRIs) can also reduce endoxifen exposure by inhibiting CYP2D6. It is thought that a reduction in endoxifen exposure reduces the efficacy of TAM treatment and increases recurrence risk. However, several recent studies contradict this hypothesis and suggest that a) there is more than one pathway to get to endoxifen, even in the presence of variant CYP2D6; and b) TAM may act through its other metabolites as well, not just endoxifen. Thus, a CYP2D6 genetic test may overly simplify our understanding of TAM metabolism and prompt clinicians to draw the wrong conclusions. As such, it would seem useful to develop an assay to directly measure each patient's unique serum metabolite levels. The ability to quantitate all the major TAM metabolites would also allow researchers to assess which metabolite level(s) most closely correlates with both recurrence and toxicity, allowing individualized patient dosing. In this regard, results from the BIG1-98 study suggest that some metabolites may be more closely associated with adverse effects than others. This finding could be clinically useful when combined with outcomes data, as poor adherence to TAM may be an unrecognized reason for a number of recurrences that could potentially be avoided by therapeutic drug monitoring (TDM) using a sensitive and specific assay. With this in mind we developed an HPLC-MS/MS method that quantitatively measures TAM and 6 of its major metabolites in a single assay. This high-throughput assay has been validated to CLIA '88 standards and is run in a high-volume commercial CLIA certified laboratory. Although some of the metabolites had been measured previously by HPLC or LC-MS/MS, this is the first assay that measures all of the major metabolites, including the newly identified norendoxifen. Results Serum from 100 women taking TAM at 20 mg/d for > 6 months was tested using this assay, and observed ranges were calculated for this patient cohort. The observed ranges from the analysis are shown in Table 1, along with the lower limit of quantitation (LLOQ) for each analyte. Table 1. Observed ranges and LLOQs for Tamoxifen and MetaboliteAnalyteLLOQ (ng/mL)Observed Range (ng/mL)Endoxifen0.40.93-43.19Tamoxifen1.512.5-233.1N-Desmethyl Tamoxifen1.53.0-374.04-Hydroxy Tamoxifen0.20.24-5.05N-Desmethyl 4'- Tamoxifen0.41.17-19.954'-Hydroxy Tamoxifen0.40.4-6.33Norendoxifen1.2 Conclusions A novel commercial assay has been developed for TAM and its metabolites, which for the first time allows physicians to use a TDM approach for their TAM-treated patients. Citation Format: Clarke NJ, Weber DM, Goldman SM, Morton JW, Lagier RJ, Birse CE, Bender RA, Waldman FM. A novel commercial LC-MS/MS assay for tamoxifen (TAM) and its major metabolites. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-09.

Detection of the BRAF V600E mutation is required for use of the BRAF inhibitor, vemurafenib, in patients with metastatic melanoma. Although the Roche Cobas 4800 BRAF V600 Mutation Test is approved, it detects primarily the single-nucleotide V600E mutation and could miss other potentially relevant V600 mutations. To assess the detection rate of the cobas assay for V600 mutations in clinical specimens, we compared the results of this assay with Sanger sequencing in 295 melanoma FFPE samples. Twenty samples were excluded because of invalid results on the cobas (n = 3), sequencing (n = 15), or both (n = 2). V600 mutations were detected by the cobas test in 96 (34.9%) of 275 samples and by Sanger sequencing in 118 (42.9%) of 275 samples. Thus, relative to Sanger sequencing, the cobas test exhibited 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Of 23 samples with positive sequencing results but negative cobas results, 21 harbored dinucleotide mutations (V600E in 6, V600K in 10, and V600R in 5); the other two involved single-nucleotide mutations (V600E and V600G). These findings indicate that the cobas assay may miss many V600 mutations in clinical specimens. In our study, the addition of Sanger sequencing for samples with negative cobas results increased the detection rate to 42.9%. This approach could help maximize the number of patients who benefit from BRAF inhibitor treatment.

Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations that indicate response to these therapies is rapidly progressing. A 34-gene next-generation sequencing (NGS) panel, developed and validated by us, was evaluated to detect additional mutations in community-based cancer specimens initially sent to our reference laboratory for routine molecular testing. Consecutive de-identified clinical specimens (n = 121) from melanoma cases (n = 31), lung cancer cases (n = 27), colorectal cancer cases (n = 33), and breast cancer cases (n = 30) were profiled by NGS, and the results were compared with routine molecular testing. Upon initial mutation testing, 20 % (24/121) were positive. NGS detected ≥1 additional mutation not identified by routine testing in 74 % of specimens (90/121). Of the specimens with additional mutations, 16 harbored mutations in National Comprehensive Cancer Network guideline genes. These various additional mutations were in gene regions not routinely covered, in genes not routinely tested, and/or present at low allele frequencies. Moreover, NGS yielded no false negatives. Overall, NGS detected mutations in 59 % of the genes (20/34) included in the panel, 75 % of which (15/20) were detected in multiple tumor types. Mutations in TP53 were found in 51 % of tumors tested (62/121). Mutations in at least one other (non-TP53) gene present in the panel were detected in 64 % of cases (77/121). This assay provides improved breadth and sensitivity for profiling clinically relevant genes in these prevalent solid tumor types.

BackgroundWell-differentiated thyroid cancer (WDTC) incidence in pediatrics is rising, most being papillary thyroid carcinoma (PTC). The objective of the study was to assess the prevalence of different mutations in pediatric WDTC and correlate the genotype with the clinical phenotype.MethodsThis is a single-center retrospective study. Thyroid tissue blocks from 42 consecutive pediatric WDTC patients who underwent thyroidectomy between 2001 and 2013 were analyzed at Quest Diagnostics for BRAF(V600E), RAS mutations (N,K,H), and RET/PTC and PAX8/PPARγ rearrangements, using validated molecular methods. Thyroid carcinomas included PTC, follicular thyroid carcinoma (FTC), and follicular variant of PTC (FVPTC).ResultsThirty-nine samples (29 females) were genotyped. The mean age at diagnosis was 14.7 years (range 7.9-18.4 years), and most were Hispanic (56.4%) or Caucasian (35.9%). The mean follow-up period was 2.9 years. Mutations were noted in 21/39 (53.8%), with both BRAF(V600E) (n = 9), and RET/PTC (n = 6) detected only in PTC. Mutations were detected in 2/5 FTC (PAX8/PPARγ and NRAS) and 3/6 FVPTC cases (PAX8/PPARγ). Of 28 PTC patients, 57.1% had mutations: 32.1% with BRAF(V600E), 21.4% with RET/PTC, and 3.6% with NRAS. Of patients with BRAF(V600E), 77.8% were Hispanic and 88.9% were >15 years, while all RET/PTC-positive patients were ≤15 years (p = 0.003). Tumor size, lymph node involvement, and distant metastasis at diagnosis (or soon after (131)I ablation) did not vary significantly based on the mutation.ConclusionsBRAF(V600E) was the most common mutation, especially in older and Hispanic adolescents. A larger, ethnically diverse pediatric cohort followed long term will enable the genotypic variability, clinical presentation, and response to therapy to be better assessed.

Objective To determine whether a next-generation sequencing (NGS) panel of 34 cancer-associated genes would cost-effectively aid in the treatment selection for patients with metastatic melanoma, compared with a single-site BRAF V600 mutation test. Methods A decision model was developed to estimate the costs and health outcomes of the two test strategies. The cost effectiveness of these two strategies was analyzed from a payer perspective over a 2-year time horizon with model parameters taken from the literature. Results In the base case, the gene sequencing panel strategy resulted in a cost of US$120,022 and 0.721 quality-adjusted life years (QALYs) per patient, whereas the single-site mutation test strategy resulted in a cost of US$128,965 and 0.704 QALYs. Thus, the gene sequencing panel strategy cost US$8943 less per patient and increased QALYs by 0.0174 per patient. Sensitivity analyses showed that, compared with the single-site mutation test strategy, the gene sequencing panel strategy had a 90.9 % chance of having reduced costs and increased QALYs, with the cost of the gene sequencing panel test having minimal effect on the incremental cost. Conclusion Compared with the single-site mutation test, the use of an NGS panel of 34 cancer-associated genes as an aid in selecting therapy for patients with metastatic melanoma reduced costs and increased QALYs. If the base-case results were applied to the 8900 patients diagnosed with metastatic melanoma in the USA each year, the gene sequencing panel strategy could result in an annual savings of US$79.6 million and a gain of 155 QALYs. Electronic supplementary material The online version of this article (doi:10.1007/s40291-015-0140-9) contains supplementary material, which is available to authorized users.

Purpose: We correlated allelic loss on chromosomes 8p, 9p and 14q with clinical outcome in locally advanced conventional renal cell carcinoma.Materials and Methods: We analyzed radical nephrectomy specimens from 72 stage P3N0 conventional renal cell carcinomas by microsatellite loss of heterozygosity (LOH) analyses directed at chromosomes 3p, 8p, 9p and 14q (2 primers per chromosome). All patients were treated with surgery only. LOH results were correlated with disease-free survival using the Kaplan-Meier method.Results: We detected LOH on chromosome 3p in 60 of 64 (94%), on chromosome 8p in 19 of 59 (32%), on 9p in 21 of 67 (31%) and on 14q in 18 of 70 (26%) informative cases. Of the 72 patients 24 (33%) had recurrence during followup. On univariate analysis patients with tumors demonstrating LOH on chromosomes 8p and 9q were at high risk for recurrence (p = 0.01). The correlation of recurrence with LOH approached significance for the individual chromosomes 8p and 9p (p = 0.10 and 0.14, respectively). No...

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

FOXP3-expressing T regulatory lymphocytes (Tregs) have been described as putative mediators of immune tolerance, and thus facilitators of tumor growth. When found in association with various malignancies, Tregs are generally markers of poor clinical outcome. However, it is unknown whether they are also associated with cancer progression. We evaluated quantitative FOXP3 expression in lymphocytes as well as in epithelial cells in a set of thirty-two breast tumors with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Tumors were stained for FOXP3 and CD3 expression and Tregs quantified by determining the ratio of colocalized FOXP3 and CD3 relative to 1) total CD3-expressing lymphocytes and 2) to FOXP3-expressing epithelial cells. The median proportion of FOXP3-expressing CD3 cells significantly increased with malignant progression from normal to DCIS to IDC components (0.005, 0.019 and 0.030, respectively; p ≤ 0.0001 for normal vs. IDC and p = 0.004 for DCIS vs. IDC). The median intensity of epithelial FOXP3 expression was also increased with invasive progression and most markedly augmented between normal and DCIS components (0.130 vs. 0.175, p ≤ 0.0001). Both Treg infiltration and epithelial FOXP3 expression were higher in grade 3 vs. grade 1 tumors (p = 0.014 for Tregs, p = 0.038 for epithelial FOXP3), but did not vary significantly with hormone receptor status, size of invasive tumor, lymph node status, or disease stage. Notably, Treg infiltration significantly correlated with epithelial up-regulation of FOXP3 expression (p = 0.013 for normal, p = 0.001 for IDC). These findings implicate both Treg infiltration and up-regulated epithelial FOXP3 expression in breast cancer progression.

e22132 Background: Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations associated with response to these targeted therapies is rapidly prog...

We correlated allelic loss on chromosomes 8p, 9p and 14q with clinical outcome in locally advanced conventional renal cell carcinoma.We analyzed radical nephrectomy specimens from 72 stage P3N0 conventional renal cell carcinomas by microsatellite loss of heterozygosity (LOH) analyses directed at chromosomes 3p, 8p, 9p and 14q (2 primers per chromosome). All patients were treated with surgery only. LOH results were correlated with disease-free survival using the Kaplan-Meier method.We detected LOH on chromosome 3p in 60 of 64 (94%), on chromosome 8p in 19 of 59 (32%), on 9p in 21 of 67 (31%) and on 14q in 18 of 70 (26%) informative cases. Of the 72 patients 24 (33%) had recurrence during followup. On univariate analysis patients with tumors demonstrating LOH on chromosomes 8p and 9q were at high risk for recurrence (p = 0.01). The correlation of recurrence with LOH approached significance for the individual chromosomes 8p and 9p (p = 0.10 and 0.14, respectively). No correlation was observed of LOH on chromosome 14q with recurrence (p = 0.42). The correlation of tumor grade with risk of relapse also approached significance (p = 0.12). On multivariate analysis LOH on chromosome 8p was a more powerful predictor of recurrence than tumor grade. When combinations of LOH on chromosomes were tested, LOH on chromosomes 8p and 9p was the most powerful predictor of recurrence (p = 0.006).LOH on chromosomes 8p or 9p may provide prognostic significance in patients with locally advanced renal cell carcinoma.

BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

7601 Background: Support for low-dose helical computed tomography (CT) screening for lung cancer in a high risk population has emerged from the National Lung Screening Trial (NLST). However, the low specificity of CT raises concerns regarding the cost and potential morbidity associated with resection of benign nodules. Non-invasive lung cancer biomarkers may serve as a useful complement to imaging, providing a simple means to further clarify the diagnosis of suspicious pulmonary nodules. SOMAmer-based chip technology was previously used to identify a panel of serological biomarkers capable of accurately classifying NSCLC. Here we assess the analytical and clinical performance of an automated assay that uses SOMAmer reagents and qPCR to simultaneously quantify a subset of these markers (n=12). Methods: Biomarker levels were measured in sera from 43 subjects with stage I-III NSCLC and 63 long-term smoker controls. qPCR results were analyzed by relative (ΔΔCT) quantitation, which uses calibrator serum and a set of normalizers. Linear mixed-effects models were fit to identify key sources of analytical variability (variance components), including plate-to-plate, within-sample, and between sample effects. Clinical performance for distinguishing NSCLC from control sera was established by a random forest predictive model. Results: Median within-sample %CV for the 12 markers was 6.7%. Variance components analyses suggest that, on average, 5.7% of the variance for a given biomarker was due to within-sample effects, 5.2% to plate-to-plate effects, and 86.2% to between-sample effects. A 10-marker random forest model exhibited an AUC [95% CI] of 0.915 [0.905, 0.924], classifying NSCLC from control sera with 79% sensitivity and 90% specificity. Conclusions: We have developed a highly reproducible automated assay designed for the clinical laboratory. Combining SOMAmer-based protein capture and qPCR-based quantification, the assay monitors the levels of 12 potential lung cancer markers. An algorithm-based model incorporating 10 of these markers showed good accuracy for distinguishing NSCLC from control sera. Further studies are warranted to evaluate the performance of the test in classifying pulmonary nodules.

8596 Background: Detection of BRAF V600 mutations is currently a prerequisite for approved use of vemurafenib in patients with metastatic melanoma. The cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a PCR-based assay approved to aid in selecting patients for vemurafenib therapy, primarily detects V600E. It is also reported to detect V600K, which has been associated with vemurafenib response as well. We compared the mutation detection rate of the cobas assay with that of Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for BRAF mutation analysis that all showed histologically-confirmed melanoma were tested. BRAF mutations were detected using both the cobas kit and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems). DNA was extracted from 5-um sections without macrodissection using the cobas DNA extraction kit (for the cobas test) or from 5-10-um sections using Agencourt extraction kits (Beckman Coulter) following macrodissection. Results: The two methods showed agreement in 104/125 (83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide mutations in 9 samples that were negative by the cobas assay. Sanger sequencing produced no results in 10 cases owing to suboptimal PCR, including 2 that were positive by the cobas assay. The cobas assay produced 2 invalid results, including 1 that was positive for V600E by Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions: Overall agreement between cobas and Sanger sequencing was 83.2%. The Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by cobas compared to the two seen by cobas but not Sanger sequencing. Thus, 16% (9/57) more patients would be identified as candidates for vemurafenib therapy using the Sanger method. [Table: see text]

2587 Background: EGFR is an oncogene product whose extracellular domain (ECD) can be either up-regulated or down-regulated in serum of patients with various cancers. In colon cancer patients with positive tissue EGFR expression, 2 EGFR ECD-targeting monoclonal antibody drugs, cetuximab (Erbitux) and panitumumab (Vectibix), are widely used chemotherapeutic agents. Patient responses to these drugs vary, and the mechanism of this variability remains unelucidated. Methods: A Slow Off-Rate Modified Aptamer (SOMAmer) targeting the EGFR ECD was selected via Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Using this SOMAmer as a capturing reagent and based on published studies ( Gold et al. PLoS ONE; Dec 7, 2010:10.1371/journal.pone.0015004), we developed a quantitative serum EGFR assay to reliably quantify EGFR in serum. Results: Recovery tests using various amounts of purified EGFR spiked into serum demonstrated a full level of EGFR. Intra and inter assay variability were tested and showed minimum variability. The detection range is 0.95 ng/ml to 600 ng/ml. Interestingly, when serum samples from patients taking cetuximab or panitumumab at the time of blood collection were tested, we observed markedly lower levels of EGFR-captured SOMAmer. ELISA assays from 2 different vendors showed normal to high levels of EGFR in these samples. We further showed that pretreatment of normal serum with either cetuximab or panitumumab can dose-dependently reduce the EGFR SOMAmer signal. The data suggest that our EGFR SOMAmer assay detects serum EGFR molecules that are unbound by cetuximab or panitumumab. Some treated patient samples had more drastic reductions in circulating serum EGFR than others. Conclusions: Various assay development parameters such as accuracy, detection range, and intra and inter assay variability showed that a SOMAmer-based assay detecting serum EGFR can be used in a clinical setting. Our data suggest that this assay can accurately measure drug-unbound EGFR in patients, which may serve as a surrogate drug efficacy indicator, and this may help physicians to adjust the drug dosage. Further studies will be necessary to investigate this possibility.

5510 Background: In 2009, the American Thyroid Association revised its guidelines for patients with thyroid nodules and differentiated thyroid cancers, recommending BRAF, RAS, RET/PTC, and PAX8-PPARγ molecular testing in patients with indeterminate fine-needle aspirate (FNA) cytology. Alterations in any of these markers in thyroid nodules are strongly associated with malignancy. We studied the prevalence of these alterations in thyroid FNA specimens in order to establish a strategy to improve disease management. Methods: DNA and RNA were extracted from thyroid FNA specimens (N=149) and tested for BRAF mutations using allele-specific PCR (V600E and K601E); for RAS (H-, K-, N-) mutations using pyrosequencing (codons 12, 13, and 61); and for RET/PTC1 and RET/PTC3 rearrangements and PAX8-PPARγ translocations using RT-PCR. Results: Overall, 90 (60.4%) of the specimens had alterations in ≥1 of the 4 molecular markers (Table). BRAF V600E mutations (54.4%) were the most prevalent mutations in papillary thyroid cancer (PTC); no K601E mutations were found. RAS mutations were found in PTC, follicular adenoma (FA), and follicular thyroid cancer (FTC) specimens; half of these mutations involved N-RAS Q61. RET/PTC rearrangements were detected in 3.5% of PTC specimens and were evenly distributed between the 2 major subtypes, RET/PTC1 and RET/PTC3. PAX8/PPARγ translocations were detected in 18.8% of FTC but not in FA, probably due to small sample size. Out of 7 indeterminate thyroid nodules, 2 had BRAF mutations and 2 had RAS mutations. The presence of the 4 markers was generally mutually exclusive; only 1 PTC specimen had concurrent RAS and RET/PTC1 alterations. Conclusions: The prevalence of BRAF, RAS, RET/PTC, and PAX8/PPARγ alterations in thyroid cancer specimens highlights the potential for targeted therapeutic strategies. The mutually exclusive pattern of alterations also suggests a hierarchical screening strategy for samples with limited availability. [Table: see text]

We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.