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CDK4/6 inhibition partly overcomes NF1 loss in T47D cells

Supplementary Table 1. Targets of The Breast NGS v1.0 capture panel. Supplementary Table 2. Gene list of NanoString nCounter{trade mark, serif} custom codeset. Supplementary Table 3. Gene list of Human Estrogen Receptor RT2 Profiler PCR Array (330231, PAHS-005ZA-24, Qiagen) Supplementary Table 4. Lines of treatment of patients with ESR1 and NF1 mutations. Supplementary Table 5. Disease free survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype. Supplementary Table 6. Advanced overall survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype

Supplementary Figure Legend

Supplementary Table 4. Haloplex re-sequencing gene mutations

Supplementary Table 5. Sanger screening of NOTCH2 exon 34

Supplementary Figure 1. Additional gene maps

Supplementary Figure 2. ABSOLUTE

Genetic profile of advanced breast cancer subtypes

NCOR expression following NF1 loss

Purpose:Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Experimental Design:Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.Results:We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant.Conclusions:Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Supplementary Table 7. Therapies regimes use to treat SMZL patients in this study

Supplementary Table 6. Somatic data from 14 matched patients

NCOR expression in NF1 mutated cancers

Genetic profile of ER positive primary breast cancers that 'switch' to triple negative recurrent advanced breast cancer

Supplementary Tables 1-3. Supplementary Table 1: Sequential samples and clonal evolution Supplementary Table 2: Haloplex gene list Supplementary Table 3: Primer sequences

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively. Clin Cancer Res; 21(18); 4174–83. ©2015 AACR.

Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas.

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised., Most testicular germ-cell tumours are exquisitely sensitive to platinum-based chemotherapies, but little is known about why 10% are resistant. Here, the authors explore the potential underlying mechanisms by probing the genomic landscape of platinum-resistant disease.

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. Experimental Design: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. Results: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Here we report the discovery of a novel Sediminibacterium sequenced from laboratory cultures of freshwater stream cyanobacteria from sites in Southern California, grown in BG11 media. A de novo genome assembly of this bacterium yielded a complete chromosomal genome on a single contig of length 3.34 Mbps with a GC content of 39.4%. A first pass annotation identified ~3000 protein coding genes, with 98% completeness when compared against all prokaryotic and cyanobacterial gene families in BUSCO. A comprehensive phylogenomic species tree reconstruction using 100 of these protein coding genes placed the novel bacterium to be sister to a previously identified Sediminibacterium, which is over 20% divergent from our novel genome.

We report a patient initially diagnosed with a triple hit high‐grade B cell lymphoma (HGBL‐TH), in which further morphologic, immunohistochemical, and next‐generation sequencing studies of subsequent specimens disclosed it to be a germinal center diffuse large B cell lymphoma (GC‐DLBCL) with BCL2/BCL6 gene translocations, PVT1‐deletion, and gain of MYC genes evolving from a previous follicular lymphoma. However, fluorescence in situ hybridization (FISH) studies with the break‐apart probe for MYC gene showed a fusion and two separated signals (red and green, respectively) leading to the interpretation of MYC gene translocation and a false diagnosis of a TH‐lymphoma, according to the recent WHO classification. Nevertheless, PVT1 deletion plus MYC gain/amplification has been described as a cause of the double‐hi transcription profile. These data highlight the need for new criteria to identify these highly aggressive lymphomas.

Longitudinal next-generation sequencing of cancer patient samples has enhanced our understanding of the evolution and progression of various cancers. As a result, and due to our increasing knowledge of heterogeneity, such sampling is becoming increasingly common in research and clinical trial sample collections. Traditionally, the evolutionary analysis of these cohorts involves the use of an aligner followed by subsequent stringent downstream analyses. However, this can lead to large levels of information loss due to the vast mutational landscape that characterizes tumor samples. Here, we propose an alignment-free approach for sequence comparison—a well-established approach in a range of biological applications including typical phylogenetic classification. Such methods could be used to compare information collated in raw sequence files to allow an unsupervised assessment of the evolutionary trajectory of patient genomic profiles. In order to highlight this utility in cancer research we have applied our alignment-free approach using a previously established metric, Jensen–Shannon divergence, and a metric novel to this area, Hellinger distance, to two longitudinal cancer patient cohorts in glioma and clear cell renal cell carcinoma using our software, NUQA. We hypothesize that this approach has the potential to reveal novel information about the heterogeneity and evolutionary trajectory of spatiotemporal tumor samples, potentially revealing early events in tumorigenesis and the origins of metastases and recurrences. Key words: alignment-free, Hellinger distance, exome-seq, evolution, phylogenetics, longitudinal.

Little information is available on the clinical significance of cancer-related genes such as KRAS, NRAS, BRAF, PIK3CA and TP53 in nonmetastatic rectal cancer. We investigated mutations of these genes in a large prospective series of locally advanced rectal cancer (LARC) patients who were recruited into two phase II trials. Mutational analyses were performed with diagnostically validated methods including polymerase chain reaction, capillary electrophoresis single-strand conformational analysis, Sanger sequencing and next-generation sequencing. Associations between single or multiple gene mutations and clinicopathological characteristics and treatment outcomes were explored. Of these 269, 210 (78%) patients were assessable. Mutations of KRAS, NRAS, BRAF, PIK3CA and TP53 occurred in 43, 9, 4, 9 and 60% of patients, respectively. Concordance between paired biopsy and resection specimens was 82% for KRAS, 95% for NRAS, 99% for BRAF, 96% for PIK3CA and 63% for TP53. TP53 mutations were associated with extramural venous invasion on baseline MRI (78% vs. 65%, p = 0.04), poor pathological tumour regression (23% vs. 36%, p = 0.05) and a trend toward a worse 5-year progression-free survival (PFS; 60% vs. 74%, HR 1.59, p = 0.06). Patients with tumours harbouring mutation of TP53 and either KRAS or NRAS (32%) had a worse 5-year PFS than those with TP53/KRAS/NRAS wild-type tumours (54% vs. 72%, HR 1.75, p = 0.02). In univariate analysis, BRAF mutation predicted poor 5-year overall survival only among patients treated without cetuximab (20% vs. 73%, HR 3.29, p = 0.03). This is one of the largest biomarker studies in a prospective, largely homogeneous, LARC population. Our findings are hypothesis generating and require validation in independent series.

Background: Patients with advanced human epidermal growth factor 2 (HER2)-positive breast cancer are at the highest risk developing breast cancer brain metastases (BCBM), which are associated with significant morbidity and mortality. The advent of HER2-directed therapy resulted in greatly improved survival outcomes and have been widely used. In this context of a better controlled systemic disease, brain metastases are emerging as a new challenge for the oncologist. The aim of this study was to determine the clinicopathologic factors associated with the prognosis of patients with HER2 positive BCBM, in the scenario of new therapies. Methods: A retrospective review of clinical data from patients who have developed HER2 positive BCBM from March 2005 to January 2018 at AC Camargo Cancer Center. Patients characteristics were collected, and overall survival (OS) , time to first HER2 positive BCBM and OS after HER2 positive BCBM were estimated by the Kaplan-Meier method. Associations between OS after BCBM and clinical variables were assessed by Cox proportional hazards regression models. Results Among 80 patients with HER2-positiveBCBM median age was 51 years (range, 24–81 years). Of the patients, 55 patients (69%) had ER-positive/HER2-positive breast cancer, and 25 (31%) had ERnegative/ HER2-positive breast cancer. The median brain metastasis-free survival period from primary breast cancer was 33.5 months .The median survival after developing brain metastasis was 28 .5 months. Patients with more than 3 brain metastases had significantly shorter overall survival(p=0,01). ). Nineteen patients (24%) underwent surgical treatment of metastasis in the brain and there was no associated with survival duration(p= 0,33). Patients treated with radiotherapy 71 (88%) had significantly better survival (p= 0,013), if the patient received stereotactic radiosurgery (53.5%) compared to total brain radiotherapy received ( 46,5%) was not associated with duration of survival (p= 0,24). . Treatment with other HER2-targeting agents after BCMB had significantly increased survival (p=0,022), including trastuzumab 53 (67%), pertuzumab 21 (26%), trastuzumab emtansine (TDM1 )= 35 (43%) and lapatinib 37 ( 46%). . In multivariate analysis, the presence of more than 3 brain metastases (p=0,005) was the sole independent prognostic factors. Conclusions: Our study indicates that HER2-positive patients with more than three brain metastases had a poor prognosis and that regardless of the use of new therapies. The role of new therapies in the management of patients with brain metastases is often not clearly defined. The best treatment strategy is not yet known and the study of prognostic factors may help to choose a better treatment sequence. Citation Format: Medeiros de Melo JC, da Costa AA, de Castro DG, Gomes AA, Guimaraes AP, Sanches SM, Diniz AC, Ribeiro AR. Prognostic factors of human epidermal growth factor 2 (HER2) positive breast cancer patients with brain metastasis in the context of all available new therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-08.

The sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) has been demonstrated to predict the response to high-dose cytarabine consolidation treatment in acute myeloid leukemia patients. Here, we evaluated SAMHD1 as potential biomarker for the response to high-dose cytarabine in mantle cell lymphoma (MCL) patients. We quantified SAMHD1 protein expression and determined the mutation status in patients of the MCL Younger and Elderly trials (n = 189), who had received high-dose cytarabine- or fludarabine-based polychemotherapy. Additionally, we quantified SAMHD1 expression in B cell lymphoma cell lines and exposed them to cytarabine, fludarabine, and clinically relevant combinations. Across both trials investigated, SAMHD1 mutations had a frequency of 7.1% (n = 13) and did not significantly affect the failure-free survival (FFS, P = .47). In patients treated with high-dose cytarabine- or fludarabine-containing regimes, SAMHD1 expression was not significantly associated with FFS or complete remission rate. SAMHD1 expression in B cell lymphoma cell lines, however, inversely correlated with their in vitro response to cytarabine as single agent (R = .65, P = .0065). This correlation could be reversed by combining cytarabine with other chemotherapeutics, such as oxaliplatin and vincristine, similar to the treatment regime of the MCL Younger trial. We conclude that this might explain why we did not observe a significant association between SAMHD1 protein expression and the outcome of MCL patients upon cytarabine-based treatment.

Somatic v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutation, present in approximately 10% of metastatic colorectal cancer (mCRC) cases, is associated with poor prognosis. Patient outcome outside of clinical trials has only been reported in small series. We report real-world data on treatment and survival for BRAF-mutated (MT) patients at a single tertiary center, compared with a matched BRAF wild type (WT) control group.All colorectal cancer patients tested for BRAF mutation, from October 2010 to November 2014 were identified. BRAF-MT mCRC cases were compared with an age and sex-matched BRAF-WT control group. Clinicopathological data were collected and survival calculated using the Kaplan-Meier method and comparisons made using Cox regression.Forty-three of 503 patients (8.5%) tested had BRAF-MT mCRC and were compared with 88 BRAF-WT controls. Median overall survival (mOS) was 18.2 months for BRAF-MT and 41.1 months for BRAF-WT mCRC patients (hazard ratio, 2.74; 95% confidence interval, 1.60-4.70; P .001). Progression-free survival for BRAF-MT and WT patients, respectively, was: 8.1 months versus 9.2 months (P = .571) first-line, 5.5 months versus 8.3 months (P = .074) second-line, and 1.8 months versus 5.6 months (P = .074) third-line. Treatment using sequential fluoropyrimidine-based doublet chemotherapy was similar between both groups. Anti-epidermal growth factor receptor (EGFR) therapy was mainly given third-line with progressive disease in 90% (n = 9 of 10) of BRAF-MT patients at first restaging.In this case-control study, the poor mOS of BRAF-MT mCRC was associated with reduced treatment benefit beyond first-line. Sequential doublet chemotherapy remains a reasonable option in appropriately selected patients. BRAF-MT patients did not benefit from anti-EGFR therapy in this study. Recruitment to clinical trials is recommended to improve outcomes in BRAF-MT mCRC.

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.

This study was supported by Cancer Research UK, AstraZeneca and Pfizer UK. Introduction: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. Methods: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. Results: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. Conclusion: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx. Publisher PDF

// Sing Y. Moorcraft 1 , Thomas Jones 2 , Brian A. Walker 1 , George Ladas 2 , Eleftheria Kalaitzaki 1 , Lina Yuan 1 , Ruwaida Begum 1 , Zakaria Eltahir 1 , Andrew Wotherspoon 1 , Angeles Montero-Fernandez 2 , Larissa S. Teixeira Mendes 1 , David Gonzalez de Castro 1 , Sanna Hulkki Wilson 1 , Paula Proszek 1 , Ye M. To 1 , Eliza Hawkes 1 , Amitesh Roy 1 , David Cunningham 1 , Sheela Rao 1 , David Watkins 1 , Naureen Starling 1 , Anne M. Bowcock 3 and Ian Chau 1 1 The Royal Marsden NHS Foundation Trust, London and Sutton, United Kingdom 2 The Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom 3 National Heart and Lung Institute, Imperial College, London, United Kingdom Correspondence to: Ian Chau, email: ian.chau@rmh.nhs.uk Keywords: colorectal cancer, heterogeneity, metastasectomy, pulmonary metastases, RAS Received: October 06, 2016 Accepted: March 29, 2017 Published: April 11, 2017 ABSTRACT This study aimed to molecularly characterise colorectal pulmonary metastases (PM) and investigate whether their molecular profiles were concordant with those of the primary tumour. Clinical data and archival formalin fixed paraffin embedded tissue samples were retrospectively collected from patients who underwent ≥ 1 pulmonary metastasectomies for colorectal cancer between 1997–2012. Primary tumour and metastatic samples were analysed using a targeted capture sequencing panel of 46 cancer-associated genes. The 5-year progression-free and overall survival rates for the 81 patients in this study were 32% (95% CI 22–42%) and 77% (95% CI 66–85%) respectively. Fifty-four patients had samples available from ≥ 1 PM, and sequencing data were successfully obtained from 33 PM from 24 patients. The most frequently mutated genes were APC (71%), KRAS (58%) and TP53 (46%). Seventy-three percent of the 15 patients with matched primary and PM samples and 6 of the 7 patients (86%) with data from ≥ 2 PM had concordant molecular profiles. The concordance for KRAS and NRAS was 100%. At our institutions, patients with resectable colorectal PM had a favourable prognosis. RAS mutations were commonly detected in PM and the molecular profiles of colorectal PM were highly concordant with the primary tumour.

Background Thyroid cancer is the most common endocrine malignancy. Some advanced disease is, or becomes, resistant to radioactive iodine therapy (refractory disease); this holds poor prognosis of 10% 10-year overall survival. Whilst Sorafenib and Lenvatinib are now licenced for the treatment of progressive iodine refractory thyroid cancer, these treatments require continuing treatment and can be associated with significant toxicity. Evidence from a pilot study has demonstrated feasibility of Selumetinib to allow the reintroduction of I-131 therapy; this larger, multicentre study is required to demonstrate the broader clinical impact of this approach before progression to a confirmatory trial. Methods SEL-I-METRY is a UK, single-arm, multi-centre, two-stage phase II trial. Participants with locally advanced or metastatic differentiated thyroid cancer with at least one measureable lesion and iodine refractory disease will be recruited from eight NHS Hospitals and treated with four-weeks of oral Selumetinib and assessed for sufficient I-123 uptake (defined as any uptake in a lesion with no previous uptake or 30% or greater increase in uptake). Those with sufficient uptake will be treated with I-131 and followed for clinical outcomes. Radiation absorbed doses will be predicted from I-123 SPECT/CT and verified from scans following the therapy. Sixty patients will be recruited to assess the primary objective of whether the treatment schedule leads to increased progression-free survival compared to historical control data. Discussion The SEL-I-METRY trial will investigate the effect of Selumetinib followed by I-131 therapy on progression-free survival in radioiodine refractory patients with differentiated thyroid cancer showing increased radioiodine uptake following initial treatment with Selumetinib. In addition, information on toxicity and dosimetry will be collected. This study presents an unprecedented opportunity to investigate the role of lesional dosimetry in molecular radiotherapy, leading to greater personalisation of therapy. To date this has been a neglected area of research. The findings of this trial will be useful to healthcare professionals and patients alike to determine whether further study of this agent is warranted. It is hoped that the development of the infrastructure to deliver a multicentre trial involving molecular radiotherapy dosimetry will lead to further trials in this field. Trial registration SEL-I-METRY is registered under ISRCTN17468602, 02/12/2015. Electronic supplementary material The online version of this article (10.1186/s12885-019-5541-4) contains supplementary material, which is available to authorized users.

Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations inOur research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non–small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology. We are enormously grateful to the Christopher's Smile charity (grant numbers CSM 002 and CSM 003)for their support and enthusiasm to provide children with more effective and less toxic targeted drugs through molecular profiling. We also thank the contribution of Children’s Cancer and Leukaemia Group (CCLG). Sí

Minimally invasive circulating free DNA (cfDNA) analysis can portray cancer genome landscapes but highly sensitive and specific genetic approaches are necessary to accurately detect mutations with often low variant frequencies. We developed a targeted cfDNA sequencing technology using novel off-the-shelf molecular barcodes for error correction, in combination with custom solution hybrid capture enrichment. Modelling based on cfDNA yields from 58 patients shows that our assay, which requires 25ng of cfDNA input, should be applicable to >95% of patients with metastatic colorectal cancer. Sequencing of a 163.3 kb target region including 32 genes detected 100% of single nucleotide variants with 0.15% variant frequency in cfDNA spike-in experiments. Molecular barcode error correction reduced false positive mutation calls by 98.6%. In a series of 28 patients with metastatic colorectal cancers, 80 out of 91 (88%) mutations previously detected by tumour tissue sequencing were called in the cfDNA. Call rates were similar for single nucleotide variants and small insertions/deletions. Mutations only called in cfDNA but not detectable in matched tumour tissue included, among others, a subclonal resistance driver mutation to anti-EGFR antibodies in theKRASgene, multiple activatingPIK3CAmutations in each of two patients (indicative of parallel evolution), andTP53mutations originating from clonal haematopoiesis. Furthermore, we demonstrate that cfDNA off-target read analysis allows the reconstruction of genome wide copy number aberration profiles from 71% of these 28 cases. This error-corrected ultra-deep cfDNA sequencing assay with a target region that can be readily customized enables broad insights into cancer genomes and evolution.

BACKGROUNDFollowing chemo-radiotherapy (CCRT) for human papilloma virus positive (HPV+) locally advanced head and neck cancer, patients frequently undergo unnecessary neck dissection (ND) and/or repeated biopsies for abnormal PET-CT, which causes significant morbidity. We assessed the role of circulating HPV DNA in identifying 'true' residual disease.METHODS We prospectively recruited test (n=55) and validation (n=33) cohorts. HPV status was confirmed by E7 RT-PCR. We developed a novel amplicon-based next generation sequencing assay (HPV16-detect) to detect circulating HPV DNA. Circulating HPV DNA levels post-CCRT were correlated to disease response (PET-CT).RESULTSIn pre-CCRT plasma, HPV-detect demonstrated 100% sensitivity and 93% specificity, and 90% sensitivity and 100% specificity for the test (27 HPV+) and validation (20 HPV+) cohorts, respectively. Thirty-six out of 37 patients (test and validation cohort) with complete samples-set had negative HPV-detect at end of treatment. Six patients underwent ND (3) and repeat primary site biopsies (3) for positive PET-CT but had no viable tumour. One patient had positive HPV-detect and positive PET-CT and liver biopsy, indicating 100% agreement for HPV-detect and residual cancer.CONCLUSIONSWe demonstrate that HPV16-detect is a highly sensitive and specific test for identification of HPV DNA in plasma at diagnosis. HPV DNA post-treatment correlates with clinical response.

Purpose: FcγR polymorphisms have been reported to enhance the immune-mediated effects of cetuximab in metastatic colorectal cancer. There are no data on the relationship between these polymorphisms and cetuximab in the early-stage setting. We performed a pharmacogenomic analysis of EXPERT-C, a randomized phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery, and adjuvant CAPOX ± cetuximab in high-risk, locally advanced rectal cancer. Experimental Design: FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms were analyzed on DNA from peripheral blood samples. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. Results: Genotyping was successfully performed in 105 of 164 (64%) patients (CAPOX = 54, CAPOX-C = 51). No deviation from the Hardy–Weinberg equilibrium or association of these polymorphisms with tumor RAS status was observed. FcγRIIa-131R (HR, 0.38; P = 0.058) and FcγRIIIa-158F alleles (HR, 0.21; P = 0.007) predicted improved progression-free survival (PFS) in patients treated with cetuximab. In the CAPOX-C arm, carriers of both 131R and 158F alleles had a statistically significant improvement in PFS (5 years: 78.4%; HR, 0.22; P = 0.002) and overall survival (OS; 5 years: 86.4%; HR, 0.24; P = 0.018) when compared with patients homozygous for 131H and/or 158V (5-year PFS: 35.7%; 5-year OS: 57.1%). An interaction between cetuximab benefit and 131R and 158F alleles was found for PFS (P = 0.017) and remained significant after adjusting for prognostic variables (P = 0.003). Conclusion: This is the first study investigating FcγRIIa and FcγRIIIa polymorphisms in patients with early-stage colorectal cancer treated with cetuximab. We showed an increased clinical benefit from cetuximab in the presence of 131R and 158F alleles. Clin Cancer Res; 20(17); 4511–9. ©2014 AACR.

Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. Methods: Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.