Tim Forshew

Analysis of circulating tumor DNA to monitor metastatic breast cancer.

BACKGROUND The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer. METHODS We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points. RESULTS Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%). CONCLUSIONS This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.).

Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA

Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1–2 years. For each case, exome sequencing was performed on 2–5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA

Sizable genomic regions were screened and low-frequency mutations were identified in circulating DNA of cancer patients using tagged-amplicon deep sequencing (TAm-Seq). Deep Sequencing Tumor DNA in Plasma Five liters of circulating blood contain millions of copies of the genome, broken into short fragments; in cancer patients, a small fraction is circulating tumor DNA (ctDNA). An even smaller number harbor mutations that affect cancer outcome. Looking for diagnostic answers in circulating DNA is a challenge, but Forshew, Murtaza, and colleagues have risen to the occasion by developing a tagged-amplicon deep sequencing (TAm-Seq) method that can amplify and sequence large genomic regions from even single copies of ctDNA. By sequencing such large regions, the authors were able to identify low-level mutations in the plasma of patients with high-grade serous ovarian carcinomas. Forshew et al. designed primers to amplify 5995 bases that covered select regions of cancer-related genes, including TP53, EGFR, BRAF, and KRAS. In plasma obtained from 38 patients with high levels of ctDNA, the authors were able to identify mutations in TP53 at allelic frequencies of 2% to 65%. In plasma samples from one patient, they also identified a de novo mutation in EGFR that had not been detected 15 months prior in the tumor mass itself. Finally, the TAm-Seq approach was used to sequence ctDNA in plasma samples collected from two women with ovarian cancer and one woman with breast cancer at different time points, tracking as many as 10 mutations in parallel. Forshew and coauthors showed that levels of mutant alleles reflected the clinical course of the disease and its treatment—for example, stabilized disease was associated with low allelic frequency, whereas patients at relapse exhibited a rise in frequency. Through several experiments, the authors were able to show that TAm-Seq is a viable method for sequencing large regions of ctDNA. Although this provides a new way to noninvasively identify gene mutations in our blood, TAm-Seq will need to achieve a more sensitive detection limit (<2% allele frequency) to identify mutations in the plasma of patients with less advanced cancers. Nevertheless, once optimized, this “liquid biopsy” approach will be amenable to personalized genomics, where the level and type of mutations in ctDNA would inform clinical decision-making on an individual basis. Plasma of cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, but sequencing of entire genes to detect cancer mutations in circulating DNA has not been demonstrated. We developed a method for tagged-amplicon deep sequencing (TAm-Seq) and screened 5995 genomic bases for low-frequency mutations. Using this method, we identified cancer mutations present in circulating DNA at allele frequencies as low as 2%, with sensitivity and specificity of >97%. We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. In another case, we identified in plasma an EGFR mutation not found in an initial ovarian biopsy. We further used TAm-Seq to monitor tumor dynamics, and tracked 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive “liquid biopsy” for personalized cancer genomics.

Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549–BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto‐inhibitory domains of BRAF and RAF1, which are replaced in‐frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low‐grade gliomas. Copyright

Mutant p53 Prolongs NF-κB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

The tumor suppressor p53 is frequently mutated in human cancer. Common mutant p53 (mutp53) isoforms can actively promote cancer through gain-of-function (GOF) mechanisms. We report that mutp53 prolongs TNF-α-induced NF-κB activation in cultured cells and intestinal organoid cultures. Remarkably, when exposed to dextran sulfate sodium, mice harboring a germline p53 mutation develop severe chronic inflammation and persistent tissue damage, and are highly prone to inflammation-associated colon cancer. This mutp53 GOF is manifested by rapid onset of flat dysplastic lesions that progress to invasive carcinoma with mutp53 accumulation and augmented NF-κB activation, faithfully recapitulating features frequently observed in human colitis-associated colorectal cancer (CAC). These findings might explain the early appearance of p53 mutations in human CAC.

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barretts esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barretts esophagus (NDBE; n = 66) and high-grade dysplasia (HGD; n = 43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barretts esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.

Targeted Therapy for BRAFV600E Malignant Astrocytoma

Purpose: Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAFV600E) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAFV600E in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAFV600E and wild-type BRAF MA. Experimental Design: BRAFV600E mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAFV600E MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo. Results: BRAFV600E mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAFV600E was identified in four instances. Using the BRAFV600E-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAFV600E mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. Conclusions: Our results indicate a 10% incidence of activating BRAFV600E among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAFV600E-specific inhibitors for treating BRAFV600E MA patients. Clin Cancer Res; 17(24); 7595–604. ©2011 AACR.

MAPK pathway activation and the origins of pediatric low-grade astrocytomas

Low‐grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment. J. Cell. Physiol. 222: 509–514, 2010.

Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1‐mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

RAF gene fusions are specific to pilocytic astrocytoma in a broad paediatric brain tumour cohort

Brain tumours are the most common solid tumour in children and are the primary cause of cancer-related death in children and young adults [4, 6]. The most prevalent childhood brain tumours are low-grade gliomas, specifically pilocytic astrocytomas (PAs, WHO Grade I) [1]. PAs are slow-growing tumours which are often cystic, and may occur sporadically or in association with the genetic disorder Neurofibromatosis type 1. Several recent studies including our own have identified novel KIAA1549–BRAF and SRGAP3–RAF1 gene fusions in the majority of PAs tested [3, 7, 8, 12]. In these fusions, the N-terminal autoinhibitory domains of the RAF proteins are replaced by those of KIAA1549 or SRGAP3, resulting in constitutive activation of the ERK/MAPK pathway. A recent study has suggested that the KIAA1549–BRAF fusion is more common in PAs originating in the cerebellum [5]. In low-grade glioma without RAF gene fusions there is increasing evidence for activation of the ERK/MAPK pathway through alternative mechanisms, such as point mutation of KRAS or BRAF [2, 11, 13]. Despite the high frequency of RAF gene fusions in PAs, they have not been investigated in other types of paediatric brain tumours. In this study, we screened a new cohort of 74 paediatric brain tumours, with a range of different pathologies, for all known KIAA1549–BRAF and SRGAP3–RAF1 fusion variants. Access to tumours and clinical data was in accordance with Local Research Ethics Committee (LREC) regulations: Great Ormond Street Hospital LREC reference number 05/Q0508/153. Tumours were classified by diagnostic criteria defined by the World Health Organization (WHO) [10]. Total RNA was extracted from fresh frozen tissue samples using the miRNeasy mini kit (Qiagen, Crawley, UK) and reverse transcribed using the SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA). KIAA1549–BRAF fusions were detected using previously described primers and techniques [3]. The primers used for detecting SRGAP3–RAF1 fusions were 50-TGG CAGTAACCTCATCACCA-30 (located in SRGAP3 exon 10) and 50-GGTTGGGTCGACAACCTTTA-30 (located in RAF1 exon 11). All fusions identified by PCR were confirmed by direct sequencing on a 3100 Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0693-y) contains supplementary material, which is available to authorized users.