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Precision Medicine in Breast Cancer: Genes, Genomes, and the Future of Genomically Driven Treatments
Abstract
Remarkable progress in sequencing technology over the past 20 years has made it possible to comprehensively profile tumors and identify clinically relevant genomic alterations. In breast cancer, the most common malignancy affecting women, we are now increasingly able to use this technology to help specify the use of therapies that target key molecular and genetic dependencies. Large sequencing studies have confirmed the role of well-known cancer-related genes and have also revealed numerous other genes that are recurrently mutated in breast cancer. This growing understanding of patient-to-patient variability at the genomic level in breast cancer is advancing our ability to direct the appropriate treatment to the appropriate patient at the appropriate time-a hallmark of "precision cancer medicine." This review focuses on the technological advances that have catalyzed these developments, the landscape of mutations in breast cancer, the clinical impact of genomic profiling, and the incorporation of genomic information into clinical care and clinical trials.
... In addition, in recent decades there has been a trend to approach tumor cells from a molecular and genetic point of view, which has dramatically improved the quality of care, especially with regard to the identification of one particular gene whose expression is altered in the genetic context of one particular form of cancer. The advent of precision medicine facilitates tumor profiling concerning genetic and genomic aspects would benefit the detection of variable types of breast cancer to appoint proper medication [8]. There are also few novel genomic assays (Oncotype DX, MammaPrint, and PAM50) from which a prognostic assessment of the risk of relapse in breast cancer can be performed at its early stage. ...
... Although breast cancer has been conventionally subtyped, molecular techniques like microarray could benefit classification of breast cancer in terms of involved genes and proteins in biological processes [13]. Improvement in genomic profiling such as different types of sequencing techniques will reduce the adverse effect of missing clinical data, i.e. it is feasible to compare the genomic or proteomic environment of breast cancer in the sense of clinical studies and in the direction of advancing in bioinformatics tools, cancer diagnosis and prediction of its response to therapy would be considerable [8]. ...
Figuring out the molecular mechanisms underlying breast cancer is essential for the diagnosis and treatment of this invasive disorder. Hence it is important to identify the most significant genes correlated with molecular events and to study their interactions in order to identify breast cancer mechanisms. Here we focus on the gene expression profiles, which we have detected in breast cancer. High-throughput genomic innovations such as microarray have helped us understand the complex dynamics of multisystem diseases such as diabetes and cancer. We performed an analysis using microarray datasets with Networkanalyst bioinformatics tool, based on a random effect model. We achieved pivotal differential expressed genes like ADAMTS5, SCARA5, IGSF10, C2orf40 that had the most down-regulation and also COL10A1, COL11A1, UHRF1 that they had most up-regulation in four-stage of breast cancer. We used CentiScape and AllegroMCODE plugins in CytoScape software in order to achieve better insight into and figure out hub genes in the protein-protein interactions network. Besides, we utilized DAVID online software to find involved biological pathways and Gene ontology, also used Expression2kinase software in order to find upstream regulatory transcription factors and kinases. In conclusion, we have found that the statistical network inference approach is useful in gene prioritization, is capable of contributing to practical network signature discovery, providing insights into the mechanisms relevant to the disease. Our study has also developed a new candidate genes, pathways, transcription factors, and kinases that may be candidates for diagnostic biomarkers and also for drug design after experimental examinations.
... Large-scale cancer genome sequencing projects have been valuable in terms of identifying the prevalent cancer driver mutations (CDMs) associated with particular types of cancers [Ciriello et al., 2013;Kandoth et al., 2013;Watson et al., 2013;Stover and Wagle, 2015]. Cancer "drivers" have been defined as genetic events associated with tumor initiation or progression [Alizadeh et al., 2015] and as somatic mutations that increase the fitness of a cell [Fisher et al., 2013]. ...
... It has been established that cancers exhibit spatial and temporal genetic diversity, meaning different mutations have been detected in different sectors of a cancer and that abundance of clonal subpopulations can evolve over time [Martelotto et al., 2014;Renovanz and Kim, 2014]. Investigations into the genetic heterogeneity of several cancers has enabled clinicians to identify patients who will benefit from particular therapies (i.e., personalized cancer treatment) and, conversely, demonstrated that mutant cell subpopulations can derail the process and drive the development of acquired resistance to therapy [Fisher et al., 2013;Burrell and Swanton, 2014;Stover and Wagle, 2015]. Given the extent of tumor heterogeneity, the prevalence of mutant subpopulations in advanced cancers and the difficulties associated with treating such cancers, it has been suggested that the development of cancer preventative modalities may be a more effective approach for reducing cancer deaths [Maresso et al., 2015]. ...
Large-scale sequencing efforts have described the mutational complexity of individual cancers and identified mutations prevalent in different cancers. As a complementary approach, allele-specific competitive blocker PCR (ACB-PCR) is being used to quantify levels of hotspot cancer driver mutations (CDMs) with high sensitivity, to elucidate the tissue-specific properties of CDMs, their occurrence as tumor cell subpopulations, and their occurrence in normal tissues. Here we report measurements of PIK3CA H1047R mutant fraction (MF) in normal colonic mucosa, normal lung, colonic adenomas, colonic adenocarcinomas, and lung adenocarcinomas. We report PIK3CA E545K MF measurements in those tissues, as well as in normal breast, normal thyroid, mammary ductal carcinomas, and papillary thyroid carcinomas. We report KRAS G12D and G12V MF measurements in normal colon. These MF measurements were integrated with previously published ACB-PCR data on KRAS G12D, KRAS G12V, and PIK3CA H1047R. Analysis of these data revealed a correlation between the degree of interindividual variability in these mutations (as log10 MF standard deviation) in normal tissues and the frequencies with which the mutations are detected in carcinomas of the corresponding organs in the COSMIC database. This novel observation has important implications. It suggests that interindividual variability in mutation levels of normal tissues may be used as a metric to identify mutations with critical early roles in tissue-specific carcinogenesis. Additionally, it raises the possibility that personalized cancer therapeutics, developed to target specifically activated oncogenic products, might be repurposed as prophylactic therapies to reduce the accumulation of cells carrying CDMs and, thereby, reduce future cancer risk.
... Genomic profiling of tumors to predict response to therapies has become an approach of precision medicine (Stover et al., 2015). Identification of biomarkers, recognition of potential targets for therapy and plausible predictions using bioinformatics platforms have accelerated the research. ...
... Genomic profiling of tumors to predict response to therapies has become an approach of precision medicine (Stover et al., 2015). Identification of biomarkers, recognition of potential targets for therapy and plausible predictions using bioinformatics platforms have accelerated the research. ...
Purpose:
miRNAs are known to be aberrantly expressed in the serum, tissue, and Peripheral Blood Mononuclear Cells (PBMC) of cancer patients and could serve as potential noninvasive diagnostic markers for breast cancer. The aim of this study was to identify the differentially expressed miRNA using next-generation sequencing (NGS) from the paired PBMC samples from breast cancer patients and age-matched healthy individuals and explore their functional significance.
Methods:
In this study, PBMCs were employed for the detection of miRNAs by NGS in locally advanced breast cancer (LABC) women of South Indian origin who were divided into three age groups, (a) 40yrs-50yrs (b) 50yrs-60yrs and (c) 60yrs-70yrs, compared with age-matched control groups.
Results:
Four miRNAs (hsa-miR-192-5p, hsa-miR-24-2-2p, hsa-miR-3609, and hsa-miR-664b-3p) were found to be differentially expressed among LABC patients compared with age matched healthy women of the South Indian population. While miR-24-2-5p, miR3609, and miR-664b-3p were down-regulated, miR-192-5p was up-regulated. Gene Ontology (GO) annotations implicated miRNA with signaling pathways in peripheral nerve synapses, glutamatergic synapse, and cell morphogenesis, all of which play a pivotal role in the manifestation of cancer.
Conclusion:
Four miRNAs- 3 (While miR-24-2-5p, miR3609, and miR-664b-3p) downregulated and one upregulated (miR-192-5p) were identified as potential biomarkers for patients with locally advanced breast cancer. These markers could be validated in studies with a larger sample size.
... 4−6 Thus, breast cancer diagnosis and treatment strategies now involve precision medicine. 7 In addition to hormone receptors, human epidermal growth factor receptor 2 (HER2), which is highly expressed in 20% of patients with breast cancer, is a crucial biomarker for breast cancer classification. 8,9 Patients with a high level of HER2 expression usually respond poorly to endocrine therapy and chemotherapy; however, a marked survival benefit is observed when these patients undergo HER2-targeted therapy. ...
... Breast cancers can be classi ed into cancer subtypes based on the expression of molecular biomarkers, which allows physicians to adjust treatment plans and response assessments for improved survival and morbidity [4][5][6]. This development in breast cancer diagnosis and treatment has led to an evolution from standard strategies to precision medicine [7]. In addition to hormone receptors, human epidermal growth factor receptor 2 (HER2), which is highly expressed in about 20% of breast cancer patients, is a crucial biomarker for breast cancer classi cation [8,9]. ...
Background: Accurate determination of human epidermal growth factor receptor 2 (HER2) expression is essential for HER2-targeted therapy. HER2 expression in a complex environment, such as in a heterogenous tumor, makes precise assessment difficult using current methods. Therefore, we developed a novel 99mTc-labeled anti-HER2-single domain antibody (99mTc-NM-02) as a molecular imaging tracer for non-invasive detection of HER2 expression and investigated its safety, radiation dosimetry, biodistribution, and tumor-targeting potential in breast cancer patients.
Methods: A lead compound (NM-02) was screened from a library of hexahistidine-tagged anti-HER2-single domain antibodies and labeled with 99mTc for the preparation of 99mTc-NM-02 tracer. Ten women with breast cancer were administered 99mTc-NM-02 at a mean dose of 458 ± 37 MBq (406−510 MBq), corresponding to 100 μg of NM-02. Whole-body and local SPECT/CT images were acquired at 1 and 2 h post-administration to investigate the tumor-targeting potential in primary and metastatic lesions. Additional images were acquired at 10 min, 3 h, and 24 h in three patients to calculate radiation dosimetry. Physical evaluation and blood analysis were performed for safety assessment.
Results: No drug-related adverse reactions occurred. The tracer mainly accumulated in the kidneys and liver with mild uptake in the spleen, intestines, and thyroid, but only background levels were observed in other organs where primary tumors and metastases typically occurred. The mean effective dose was 6.56 × 10⁻³ mSv/MBq, and tracer uptake was visually observed in primary tumors and metastases. Owing to the fast clearance of the tracer, we were able to sufficiently discern uptake over normal background in both primary lesions and metastases within 2 h after injection. A maximal standard uptake value of 1.5 could be a reasonable cutoff for determining HER2 positivity using SPECT/CT imaging.
Conclusions: Our 99mTc-NM-02 tracer can be safely used for imaging in breast cancer patients with reasonable radiation doses, favorable biodistribution and imaging characteristics. 99mTc-NM-02 SPECT imaging may provide an accurate and non-invasive method to detect HER2 status in breast cancer patients.
Trial registration: ClinicalTrials.gov, NCT04040686. Registered 30 July 2019. https://clinicaltrials.gov/ct2/show/NCT04040686.
... The personalized approach can favorably change the prognosis of many women suffering from breast cancer (BC), especially in case of the most difficult to treat BC subtypes (e.g., the triple-negative BC and HER2-positive BC) [11,12,13,14]. Approximately 15-20% of BCs overexpress HER2 that is related to aggressive tumor behavior and decreased survival [15,16]. ...
Precision medicine considers specific biological characteristics of each individual patient to tailor diagnostic and therapeutic strategies to a given patient. This approach is particularly important for a growing number of patients with malignancies. Currently, some unique biological properties in the terms of different “omics” platforms (e.g., genomics, proteomics, transcriptomics, metabolomics, epigenomics, and pharmacogenomics) have been introduced to precision medicine. In addition, specific personal characteristics of the patients have been described as personomics. It should be highlighted that personomics include an individual patient’s personality type, set of personal values, priorities, preferences, health-related beliefs, goals, economical status, and different life circumstances, which influence when and how a certain disease (e.g., breast cancer (BC)) can be manifested in a given person. As a consequence, personomics are considered to be an innovative clinical tool that is crucial for making a connection between the existing and emerging, more individualized model of medical care. This is particularly important among patients suffering from the most difficult to treat cancers (e.g., BC subtypes, such as the triple-negative BC (TNBC), and the human epidermal growth factor receptor 2 (HER2)-positive BC). This mini-review addresses some research concepts in personalized medicine, focusing on personomics, which apply individualized data of the patient to the therapeutic plan. In this light, personomics can facilitate the transition from standard medical treatment to personalized medical management of individual women with BC.
... One of the mechanisms that causes resistance of endocrine therapy are mutations on the estrogen receptor-α (ESR1) gene that render the ER from constitutive activation. From the perspective of cancer genomics, luminal A subtypes are shown to have the most mutated genes, with the most frequent in PIK3CA, followed by MAP3K1, GATA3, TP53, CDH1 and MAP2K4 [21] . Everolimus, a mTOR inhibitor, has been shown to increase the efficacy of endocrine therapy and overcome resistance to endocrine therapies. ...
Breast cancer (BC) is a malignancy with the highest incidence in women. Great progress has been made in research related to traditional precision medicine for BC. However, many reports have suggested that patients with BC have not benefited a lot from such progress. Thus, we analyze traditional precision medicine strategies for BC, sum up their limitations and challenges, and preliminarily propose future orientations of precision medicine strategies based on a database on drug reaction of patients with BC. According to related research, traditional precision medicine strategies for BC, which are based on molecular subtypes, perform pertinent treatments, new drug research and development according to molecular typing results. Nevertheless, these strategies still have some deficiencies. First, there are very few patients with each molecular subtype, the match ratio of drugs is low. Second, these strategies can not solve the problem of poor drug sensitivity resulting from heterogeneity. The main strategy we put forward in the present paper is based on patients’ varying drug reactions. Focusing on treating existing patients and maximizing the utilization of existing drugs, it is expected to not have deficiencies of traditional precision medicine for BC, including low match rate and poor therapeutic efficacy arising from tumor heterogeneity of BC.
... TNBCs are characterized by the absence of the estrogen and progesterone hormone receptors as well as HER2 overexpression, and their treatment remains a challenge due to the lack of defined targeted therapies [28,29]. These tumors exhibit molecular heterogeneity and distinct gene expression signatures, which further indicate the need for appropriately tailored patient-specific targeted therapy [30,31]. The current standard of care for TNBCs is chemotherapy-based regimes. ...
Protein methyltransferase SUV39H2 was reported to methylate histone H2AX at lysine 134 and enhance the formation of phosphorylated H2AX (γ-H2AX), which causes chemoresistance of cancer cells. We found that a series of imidazo[1,2-a]pyridine compounds that we synthesized could inhibit SUV39H2 methyltransferase activity. One of the potent compounds, OTS193320, was further analyzed in in vitro studies. The compound decreased global histone H3 lysine 9 tri-methylation levels in breast cancer cells and triggered apoptotic cell death. Combination of OTS193320 with doxorubicin (DOX) resulted in reduction of γ-H2AX levels as well as cancer cell viability compared to a single agent OTS193320 or DOX. Further optimization of inhibitors and their in vivo analysis identified a compound, OTS186935, which revealed significant inhibition of tumor growth in mouse xenograft models using MDA-MB-231 breast cancer cells and A549 lung cancer cells without any detectable toxicity. Our results suggest that the SUV39H2 inhibitors sensitize cancer cells to DOX by reduction of γ-H2AX levels in cancer cells, and collectively demonstrate that SUV39H2 inhibition warrants further investigation as a novel anti-cancer therapy.
... Indeed, the genetic variability that underlies, for example, breast cancer is starting to "direct the appropriate treatment to the appropriate patient at the appropriate time" -so-called 'precision medicine'. 2 More recently, genetic analysis helped delineate five subtypes of 'adult-onset diabetes' (see Table 1), which might represent "a first step towards precision medicine in diabetes". 3 "This paper has helped to start a vital conversation about how we categorise types of diabetes. ...
The recently published Lancet Diabetes and Endocrinology paper that identifies five subtypes of adult‐onset diabetes associated with differing risk of complications has sparked new debate on the classification of diabetes and may help bring precision medicine a step closer.
... ER/PR/HER2 status is known to affect patient survival (with socalled triple negative patients having the worst survival) and helps physicians select appropriate treatment regimens [9]. A host of targeted therapeutic options would now be available to be used based on the genomic profiling of this woman's tumor [10]. The physician's recommendation about systemic chemotherapy would be informed by the 21-gene recurrence score assay that may be used to stratify the risk of recurrence of this woman's type of early-stage, ER-positive breast cancer [11]. ...
Clinical practice guidelines have been developed for many common conditions based on data from randomized controlled trials. When medicine is informed solely by clinical practice guidelines, however, the patient is not treated as an individual, but rather a member of a group. Precision medicine, as defined herein, characterizes unique biological characteristics of the individual or of specimens obtained from an individual to tailor diagnostics and therapeutics to a specific patient. These unique biological characteristics are defined by the tools of precision medicine: genomics, proteomics, metabolomics, epigenomics, pharmacogenomics, and other "-omics." Personalized medicine, as defined herein, uses additional information about the individual derived from knowing the patient as a person. These unique personal characteristics are defined by tools known as personomics which takes into account an individual's personality, preferences, values, goals, health beliefs, social support network, financial resources, and unique life circumstances that affect how and when a given health condition will manifest in that person and how that condition will respond to treatment. In this paradigm, precision medicine may be considered a necessary step in the evolution of medical care to personalized medicine, with personomics as the missing link.
... Somatic mutations, in concert with information about copy number alterations, DNA methylation and gene expression patterns allow the specification of therapeutic regimes, targeting crucial molecules on which the survival and the proliferation of cancer cells depend. The appropriate choice of treatment of individual patients led to a new precision in cancer medicine (Stover and Wagle, 2015;Friedman et al., 2015a;Iorio et al., 2016). ...
... Since the majority of the potentially beneficial effects of melatonin have been described in estrogen-responsive breast cancer, the present study searched information published on 84 genes known to be dysregulated during breast carcinogenesis (corresponding to the genes included in The Human Breast Cancer RT² Profiler PCR array; Qiagen GmbH, Hilden, Germany). These genes encode proteins implicated in signal transduction, angiogenesis, proteolysis, cell cycle and apoptosis (154). The present study also reviewed recently published articles associating melatonin with chemotherapeutic agents (135)(136)(137)(142)(143)(144)(145)(146)(147)(148)(149). ...
... Promising results have also been found in immunological biomarkers [66,67]. However, although several biomarker studies have been conducted successfully using genetic or molecular analysis to predict treatment outcome, this approach has only been successfully validated for clinical routine in estrogen receptor positive, lymph node negative breast cancer patients [68][69][70]. Only these two methods were able to successfully add clinical predictive data to the established clinical characteristics [71]. ...
Background
Targeted anti-HER2 therapy has greatly improved the prognosis for many breast cancer patients. However, treatment for HER2 negative disease is currently still selected from a multitude of untargeted chemotherapeutic treatment options. A predictive test was developed using patient-derived spheroids to identify the most effective therapy for patients with HER2 negative breast cancer of all stages, for clinically relevant subgroups, as well as individual patients.
Methods
Tumor samples from 120 HER2 negative patients obtained through biopsy or surgical excision were tested in the breast cancer spheroid model using scaffold-free cell culture. Similarly, spheroids were also generated from established HER2 negative breast cancer cell lines T-47D, MCF7, HCC1143, and HCC1937 to compare treatment efficacy of heterogeneous cell populations from patient tumor tissue with homogeneous cell lines. Spheroids were treated in vitro with guideline-recommended compounds. Treatment mediated impact on cell survival was subsequently quantified using an ATP assay.
Results
Differences were observed in the metabolic activity of the untreated spheroids, whereby cell lines consistently achieved higher values compared to tissue spheroids (p < 0.001). A higher number of cells per spheroid correlated with a higher basal metabolic activity in tissue-derived spheroids (p < 0.01), while the opposite was observed for cell line spheroids (p < 0.01). Recurrent tumors showed a higher mean vitality (p < 0.01) compared to primary tumors. Except for taxanes, treatment efficacy for most tested compounds differed significantly between breast cancer tissue spheroids and breast cancer cell lines. Overall a high variability in treatment response in vitro was seen in the tissue spheroids regardless of the tested substances. A greater response to anthracycline/docetaxel was observed for hormone receptor negative samples (p < 0.01). A higher response to 5-FU (p < 0.01) and anthracycline (p < 0.05) was seen in high grade tumors. Smaller tumor size and negative lymph node status were both associated with a higher treatment efficacy to anthracycline treatment combined with 5-FU (cT1/2 vs cT3/4, p = 0.035, cN+ vs cN−, p < 0.05).
Conclusions
The tissue spheroid model reflects current guideline treatment recommendations for HER2 negative breast cancer, whereas tested cell lines did not. This model represents a unique diagnostic method to select the most effective therapy out of several equivalent treatment options.
PURPOSE
Tumor genomic testing (TGT) has become increasingly adopted as part of standard cancer care for many cancers. Despite national guidelines around patient education before TGT, available evidence suggests that most patients' understanding of genomics remains limited, particularly lower-income and minority patients, and most patients are not informed regarding potential incidental germline findings.
METHODS
To investigate and address limitations in patient understanding of TGT results, a Plan-Do-Study-Act (PDSA) approach is being used to assess needs, identify opportunities for improvement, and implement approaches to optimize patient education. We reviewed published guidelines related to pre-TGT provider-patient education and to identify key points (Plan). A provider quality improvement survey was completed (Do), which highlighted inconsistency in pre-TGT discussion practice across providers and minimal discussion with patients regarding the possibility of incidental germline findings.
RESULTS
Patient focus groups and interviews (N = 12 patients) were completed with coding of each transcript (Study), which revealed themes including trouble differentiating TGT from other forms of testing, yet understanding that results could tailor therapy. The integration of data across this initial PDSA cycle identified consistent themes and opportunities, which were incorporated into a patient-directed, concise animated video for pre-TGT education (Act), which will form the foundation of a subsequent PDSA cycle. The video addresses how TGT may/may not inform treatment, the process for TGT using existing tissue or liquid biopsy, insurance coverage, and the potential need for germline genetics follow-up because of incidental findings.
CONCLUSION
This PDSA cycle reveals key gaps and opportunities for improvement in patient education before TGT.
The normal mammary gland’s development, maintenance, and function depend on a complex signal transduction system orchestrated by integrins, growth factors, and steroid hormones. Changes in these cell signal transduction pathways have been associated with breast cancer development and progression. Despite technological advances in diagnostic methods and molecular classification, breast cancer morbidity and mortality rates remain unchanged. A significant number of patients die due to the formation of metastases, resistance to treatment, and disease recurrence. Thus, one of the main objectives of research in breast cancer is the identification and characterization of new biomarkers, which can be used as prognostic and predictive factors of response or indicators of therapeutic resistance, preventing disease recurrence. Par-4 (Prostate Apoptosis Response 4; also known as PAWR, pro-apoptotic WT1 regulator) is a tumor suppressor gene that encodes a pro-apoptotic protein expressed ubiquitously among the various tissue types, and for which altered expression patterns are already described in many types of cancers, including breast cancer. In this chapter, we review the possible roles of Par-4 in breast cancer, providing insights into the potential role of this tumor suppressor gene as a novel prognostic and predictive biomarker of this complex and heterogeneous disease.
Prolactin (PRL) is a key hormone involved in canine mammary development and tumorigenesis. In this study, the influence of a single nucleotide polymorphism (SNP) in the PRL gene (rs23932236) on the clinicopathological parameters and survival of dogs with canine mammary tumors (CMTs) was investigated. A total of 206 female dogs with spontaneous mammary tumors were enrolled in this study and circulating blood cells were genotyped. This specific SNP was associated with larger size (>3 cm diameter) for malignant tumors ( P = .036), tumors with infiltrative/invasive growth pattern ( P = .010), vascular invasion ( P = .006), and lymph node metastasis ( P = .004). Carriers of the variant allele had a shorter overall survival compared to the wild-type population with an overall survival of 18.7 months and 22.7 months, respectively ( P = .004). These findings suggest that SNP rs23932236 of canine PRL gene may be used as an indicator for the development of clinically aggressive forms of CMTs.
The frequency of cancer presents an increasing trend in humans and companion animals, and despite recent advances in diagnosis and treatment, it remains a major cause of morbidity and mortality in human and veterinary medicine. The epidemiological and clinicopathological similarities between spontaneous tumours of companion animals and their human counterparts make them suitable natural models for human cancer research. Moreover, the faster progression of cancer in dogs and cats in comparison with humans, associated with the shorter life span of companion animals, enables faster data retrieval than in human malignancies. Furthermore, the health effects associated with exposure to environmental hazardous materials, including cancer, occur similarly in companion animals and humans; consequently, in an epidemiological context, dogs and cats can also be useful as sentinels of human malignancies. For these reasons, comparative oncology, which can be defined as the study of spontaneous cancers in animals as models for human disease, has gained increasing importance over the last decades. Breast cancer represents the most prevalent cancer among women worldwide and the leading cause of cancer-related mortality in women. Mammary gland tumours are also among the most frequent tumours in female dogs and cats. Canine and feline mammary tumours present similar incidence, relative age of onset, risk factors, biological behaviour, metastatic pattern, histological, molecular, and genetic features, and response to therapy to human breast cancer; thus, they are recognized as suitable natural models for human breast cancer studies. The comparative “One Health” approach allows advances in knowledge of the diseases in order to obtain an improvement in clinical outcomes for affected humans and animals.
E‐cadherin is a cell adhesion molecule that participates in several cellular processes that guarantee the maintenance of structural and functional integrity of epithelial tissues. E‐cadherin plays an important role in mammary carcinogenesis and various studies have demonstrated the effect of CDH1 genetic variation in risk, progression and biological behavior of human breast cancer. Although there are some recognized genetic variations in canine CDH1 gene, their influence in canine mammary tumor development and progression has not been previously evaluated. In this study, we aim to assess the influence of CDH1 SNPs rs850805755, rs852280880 and rs852639930 in the risk, clinicopathological features and clinical outcome of canine mammary tumors. A case‐control study was conducted involving 206 bitches with mammary tumors and 161 bitches free of mammary neoplasia. CDH1 SNPs rs850805755 and rs852280880 were associated with a decreased risk and a later onset of mammary tumor development. Furthermore, these SNPs were related to the development of small size carcinomas, of low histological grade and low nuclear pleomorphism. SNP rs852639930 was associated with the development of small size tumors with a non‐infiltrative, non‐invasive growth pattern. Data from the present investigation demonstrate that these CDH1 genetic variants could have a protective role in canine mammary tumors, by being associated with low risk of tumor development, delayed onset of the disease and less aggressive clinicopathological features.
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The mechanistic target of rapamycin (mTOR) is a fundamental component of a signaling pathway that is involved in the pathogenesis of breast cancer via different mechanisms. This pathway is functionally linked with a number of small nucleolar RNA host genes (SNHGs). In the present project, we have searched for the expression quantitative trait loci (eQTLs) within SNHGs that are possibly involved in the pathogenesis of breast cancer. Following this in silico step, we have assessed expression levels of mTOR and four SNHGs in malignant and nonmalignant samples obtained from 80 patients with breast cancer. We also genotyped rs4615861 of SNHG3 and rs3087978 of SNHG5 in the peripheral blood of patients. SNHG12 expression was not detected in any of the assessed malignant or nonmalignant tissues. So this gene was excluded from further steps. Expression of mTOR and other three long noncoding RNAs (lncRNAs) were significantly increased in the malignant tissues compared with the nonmalignant tissues. When classifying patients into down‐/upregulation categorized based on the transcript levels of each gene in malignant tissue versus nonmalignant tissues, we noticed associations between expression of SNHG1 and stage (p = 0.03), expression of SNHG5 and grade (p = 0.05), as well as between expression of SNHG3 and history of oral contraceptive use (p = 0.04). We also detected higher levels of SNHG3 expression in estrogen receptor/progesterone receptor (ER/PR) negative tumors compared with the ER/PR positive tumors (p = 0.003 and p = 0.01, respectively). Moreover, there was a trend toward higher expression of this lncRNA in HER2‐positive tumors compared with the HER2‐negative ones (p = 0.07). Combination of transcript levels of all genes could differentiate malignant tissues from nonmalignant tissues with the diagnostic power of 69% (p = 0.0001). The rs3087978 was associated with the expression of mTOR in malignant tissues in a way that TT and TG genotypes were associated with the higher and lower levels of expressions, respectively (p = 0.01). The current study underscores the significance of SNHGs in the pathogenesis of breast cancer.
As NF‐κB signaling pathway is constitutively activated in lung cancer, targeting NF‐κB has a potential for the treatment. EF24 has been proved to be a NF‐κB inhibitor with good anti‐tumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF‐κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF‐κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF‐κB. Taken together, it was suggested that 13d was a potent anti‐tumor agent.
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Breast cancer is the most common cancer among women and represents one of the top five leading causes of cancer-related mortality. Inherited and acquired genetic mutations as well as epigenetic aberrations are known to be important contributors to the development and progression of breast cancer. Recent developments in high-throughput technologies have increased our understanding of the molecular changes in breast cancer, leading to the identification of distinctive genetic and epigenetic modifications in different breast cancer molecular subtypes. These genetic and epigenetic changes in luminal A, luminal B, ERBB2/HER2-enriched, basal-like, and normal-like breast cancer subtypes are discussed in this chapter. Furthermore, recent epigenome studies provided more information about further stratification of breast cancer subtypes, with essential role in the appropriate diagnosis and treatment of breast cancer. Thus, the inclusion of both genetic and epigenetic information in breast cancer clinical care could provide critical scientific base for precision medicine in breast cancer.
The enzyme catechol‐o‐methyltransferase (COMT) is responsible for inactivation of catechol estrogens, which are well‐recognized carcinogenic metabolites. Two single‐nucleotide polymorphisms (SNPs) in canine COMT gene were previously associated with the age of onset of mammary tumours and with the clinical progression of the disease. However, no information is available regarding the impact of other known SNPs in COMT gene in canine mammary tumours. The aim of the present study is to evaluate the influence of COMT SNP in clinicopathological features and outcome of canine mammary tumours. A case series study was conducted involving 155 non‐neutered bitches with mammary tumours submitted to follow‐up for 24 months after surgery. Three genotypes were considered: Genotype 1 (rs853046495); Genotype 2 (rs23350589, rs23322686, rs23336579, and rs852564758); Genotype 3 (rs851328636 and rs853133060). Genotype 1 was associated to low degree of tubular differentiation. Genotype 2 was related to the development of intermediate/high‐histological‐grade carcinomas and to vascular invasion. Genotype 3 was associated to reduced nuclear pleomorphism and well‐differentiated carcinomas. Data from the present investigation allowed the identification of COMT genetic profiles associated with pathological features of mammary tumours that constitute relevant prognostic factors. The assessment of the COMT genotypes may represent a helpful tool in the clinical management of canine mammary tumours, assisting in the selection of individualized preventive and therapeutic strategies, according to the animals' genetic profile.
Expert curation at present remains the only reliable solution for interpretation of mutation information in the clinical practice of oncology.
Precision medicine refers to the idea of delivering the right treatment to the right patient at the right time, usually with a focus on a data-centred approach to this task. In this perspective piece, we use the term "precision healthcare" to describe the development of precision approaches that bridge from the individual to the population, taking advantage of individual-level data, but also taking the social context into account. These problems give rise to a broad spectrum of technical, scientific, policy, ethical and social challenges, and new mathematical techniques will be required to meet them. To ensure that the science underpinning "precision" is robust, interpretable and well-suited to meet the policy, ethical and social questions that such approaches raise, the mathematical methods for data analysis should be transparent, robust and able to adapt to errors and uncertainties. In particular, precision methodologies should capture the complexity of data, yet produce tractable descriptions at the relevant resolution while preserving intelligibility and traceability, so that they can be used by practitioners to aid decision-making. Through several case studies in this domain of precision healthcare, we argue that this vision requires the development of new mathematical frameworks, both in modelling and in data analysis and interpretation.
Targeting specific mutations that have arisen within a tumour is a promising means of increasing the efficacy of treatments, and breast cancer is no exception to this new paradigm of personalised medicine. Traditional DNA sequencing methods used to characterise clinical cancer specimens and impact treatment decisions are highly sensitive, but are often limited in their scope to known mutational hot spots. Next-generation sequencing (NGS) technologies can also test for these well-known hot spots, as well as identifying insertions and deletions, copy number changes such as ERBB2 (HER2) gene amplification, and a wide array of fusion or rearrangement events. By rapidly analysing many genes in parallel, NGS technologies can make efficient use of precious biopsy material. Comprehensive genomic profiling (CGP) by NGS can reveal targetable, clinically relevant genomic alterations that can stratify tumours by predicted sensitivity to a variety of therapies, including HER2- or MTOR-targeted therapies, immunotherapies, and other kinase inhibitors. Many clinically relevant genomic alterations would not be identified by IHC or hotspot testing, but can be detected by NGS. In addition to the most common breast carcinoma subtypes, rare subtypes analysed with CGP also harbour clinically relevant genomic alterations that can potentially direct therapy selection, illustrating that CGP is a powerful tool for guiding treatment across all breast cancer subtypes.
A lipid-conjugated, estrogenic derivative molecule (ESC8) that, in compare to other estrogenic molecules encouraged cell death in both ER-positive and ER-negative breast cancer cells. A rapid and highly sensitive assay method has been developed and validated for the estimation of a ESC8 in rat plasma using liquid chromatography coupled with mass spectrometry under a positive-ion mode with electrospray ionization. The sample process includes using methanol for precipitation of ESC8 and dextromethorphan (internal standard, IS) from plasma. Chromatographic separation was achieved with methanol: water: formic acid (70:30:0.1 % v/v/v) pumped at a flow rate of 0.3 ml/min and a C18 column (50 × 2.1 mm i.d.,1.7 µm particle size) with a total run time of 5 min. The m/z ions monitored were 568.5 and 272.1 for ESC8 and IS, respectively. The lower limit of quantitation achieved was 1.08 ng/ml and linearity was observed from 5 to 500 ng/ml. The intra- and inter-day precision was less than 4%. The proposed method was successfully applied to a preliminary pharmacokinetic study of ESC8 liposomal formulation following an intraperitoneal administration of 3.67 mg/kg in rats. The concentrations of ESC8 in plasma were quantifiable up to 36 h and Cmax of ESC8 was found to be 110.72 ng/ml, AUC 1625.23 ng/ml*h and T1/2 11.72 h.
Big-data-based edge biomarker is a new concept to characterize disease features based on biomedical big data in a dynamical and network manner, which also provides alternative strategies to indicate disease status in single samples. This article gives a comprehensive review on big-data-based edge biomarkers for complex diseases in an individual patient, which are defined as biomarkers based on network information and high-dimensional data. Specifically, we firstly introduce the sources and structures of biomedical big data accessible in public for edge biomarker and disease study. We show that biomedical big data are typically 'small-sample size in high-dimension space', i.e. small samples but with high dimensions on features (e.g. omics data) for each individual, in contrast to traditional big data in many other fields characterized as 'large-sample size in low-dimension space', i.e. big samples but with low dimensions on features. Then, we demonstrate the concept, model and algorithm for edge biomarkers and further big-data-based edge biomarkers. Dissimilar to conventional biomarkers, edge biomarkers, e.g. module biomarkers in module network rewiring-analysis, are able to predict the disease state by learning differential associations between molecules rather than differential expressions of molecules during disease progression or treatment in individual patients. In particular, in contrast to using the information of the common molecules or edges (i.e.molecule-pairs) across a population in traditional biomarkers including network and edge biomarkers, big-data-based edge biomarkers are specific for each individual and thus can accurately evaluate the disease state by considering the individual heterogeneity. Therefore, the measurement of big data in a high-dimensional space is required not only in the learning process but also in the diagnosing or predicting process of the tested individual. Finally, we provide a case study on analyzing the temporal expression data from a malaria vaccine trial by big-data-based edge biomarkers from module network rewiring-analysis. The illustrative results show that the identified module biomarkers can accurately distinguish vaccines with or without protection and outperformed previous reported gene signatures in terms of effectiveness and efficiency.
The authors review the principles of systemic therapy in breast cancer. They analyze the degree of treatment individualization in our current approach. New technologies allow the detection of genomic alterations in cancer cells. Unfortunately, we do not know yet how to best use this knowledge for routine patient care. Most genomic alterations are rare events complicating further drug development. Temporal and spatial heterogeneity in tumors also has to be taken into account. An intense international collaboration is ongoing to try and demonstrate that precision medicine will really improve treatment outcome.
Cytotoxic chemotherapy remains the systemic therapy of choice for triple-negative breast cancer (TNBC), a poor-prognosis subtype of breast cancer. Growing data focusing on TNBC provides an opportunity to assess if we can tailor adjuvant chemotherapy based on patient and tumor characteristics. The standard of care for moderate-to-high risk TNBC remains a sequential anthracycline-taxane combination, with the potential for shorter and less toxic regimens in stage I disease. Platinums are promising in the neoadjuvant and metastatic settings but we await long-term outcome data before incorporation into standard regimens in the adjuvant setting. Specific subgroups within TNBC, such as BRCA mutation carriers, require special attention, and the role of platinums in these patients warrants further consideration. There is hope that in the future, further subdividing TNBC by gene expression profile, mutation, immune infiltrate, and others will reveal novel susceptibilities.
Copyright © 2015. Published by Elsevier Ltd.
Aim: Chromosomal rearrangements of the ROS1 receptor tyrosine kinase gene define a distinct molecular subset of non-small cell lung cancer (NSCLC), which may be susceptible to therapeutic ROS1 inhibition. Crizotinib is a small molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1 and c-MET.
Methods: Fifty patients with advanced NSCLC harboring a ROS1 rearrangement were enrolled into an expansion cohort of the global phase 1 study of crizotinib. Local ROS1 testing was used, including break-apart fluorescence in situ hybridization (FISH) in 47 of 50 cases. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily, and assessed for safety and response to therapy. The primary endpoint was objective response rate (ORR). Duration of response (DR), progression-free survival (PFS), and overall survival (OS) were estimated using the Kaplan-Meier method. ROS1 fusion partners (if tissue was available) were identified using next generation sequencing (NGS) or reverse-transcriptase-polymerase-chain reaction (RT-PCR) assays.
Results: At the time of data cutoff, median follow-up of the 50 ROS1 patients was 16.1 months. The majority of patients (86%) had received at least one prior line of standard therapy for advanced NSCLC. The ORR was 70% (95% CI, 55%, 82%), with 3 complete responses and 32 partial responses. Median DR was 75.9 weeks (95% CI, 61.6, NR). Median PFS was 19.2 months (95% CI, 14.6, NR), with 24 patients (48%) still in follow-up for PFS. Median OS has not been reached; 1-year OS was 85% (95% CI, 71%, 93%). Among 24 tumors tested (21 with NGS and 3 with RT-PCR), 7 different ROS1 fusion partners were identified, including 5 known and 2 novel partner genes. No correlation was observed between type of ROS1 rearrangement and clinical response to crizotinib. The safety profile of crizotinib in ROS1 patients was similar to that seen in patients with ALK-rearranged NSCLC.
Conclusions: Crizotinib exhibits robust antitumor activity in patients with advanced ROS1-rearranged NSCLC, with an ORR of 70% and a median PFS of 19.2 months. Responses were durable with a median DR of 75.9 weeks. In this trial ROS1 defines an additional distinct population of NSCLC patients for whom crizotinib is highly effective.
Disclosure: A. Shaw: Consultant/SAB: Pfizer, Novartis, Ariad, Chugai, Genentech, Ignyta; S.I. Ou: Advisory boards: Pfizer; Corporate-sponsored research: Pfizer; Y. Bang: Advisory boards: Pfizer; Corporate-sponsored research: Pfizer; B. Solomon: Advisory boards: Pfizer, Novartis, Roche, Clovis Oncology, AstraZeneca, Merck, BMS, Lilly; G.J. Riely: Advisory boards: Ariad, Celgene, Mersana; Corporate-sponsored research: Novartis, Pfizer, Millennium, Roche, GSK, Merck; M. Varella-Garcia: Advisory board: Exelixis; Corporate-sponsored research: Abbott Molecular; Other substantive relationships (Speaker engagement in educational seminars): Abbott Molecular; D. Costa: Other substantive relationships (Consultancy): Pfizer; R. Doebele: Advisory board: Pfizer, Boehringer-Ingelheim; Corporate-sponsored research: Pfizer, Mirati Therapeutics; L.P. Le: Stock ownership: Enzymatics; Other substantive relationships (Consulting with compensation): Enzymatics; Z. Zheng: Stock ownership: Enzymatics; P. Stephenson: Other substantive relationships (Consultancy): Pfizer; S.M. Shreeve: Stock ownership: Johnson and Johnson, Pfizer; Other substantive relationships (Employment): Janssen Pharmaceutical Companies of Johnson and Johnson; L. Tye: Employment and stock ownership: Pfizer; J. Christensen: Stock ownership: Pfizer; K. Wilner: Employment, stock ownership, advisory board and corporate-sponsored research: Pfizer; A. Iafrate: Advisory boards: Chugai, Constellation, Enzymatics; Stock ownership: Enzymatics. All other authors have declared no conflicts of interest.
Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.
Background:
Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.
Methods:
We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays.
Results:
The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.
Conclusions:
In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).
Metastatic breast cancer is one of the leading causes of cancer-related mortality among women in the Western world. To date most research efforts have focused on the molecular analysis of the primary tumour to dissect the genotypes of the disease. However, accumulating evidence supports a molecular evolution of breast cancer during its life cycle, with metastatic lesions acquiring new molecular aberrations. Recognising this critical gap of knowledge, the Breast International Group is launching AURORA, a large, multinational, collaborative metastatic breast cancer molecular screening programme. Approximately 1300 patients with metastatic breast cancer who have received no more than one line of systemic treatment for advanced disease will, after giving informed consent, donate archived primary tumour tissue, as well as will donate tissue collected prospectively from the biopsy of metastatic lesions and blood. Both tumour tissue types, together with a blood sample, will then be subjected to next generation sequencing for a panel of cancer-related genes. The patients will be treated at the discretion of their treating physicians per standard local practice, and they will be followed for clinical outcome for 10 years. Alternatively, depending on the molecular profiles found, patients will be directed to innovative clinical trials assessing molecularly targeted agents. Samples of outlier patients considered as 'exceptional responders' or as 'rapid progressors' based on the clinical follow-up will be subjected to deeper molecular characterisation in order to identify new prognostic and predictive biomarkers. AURORA, through its innovative design, will shed light onto some of the unknown areas of metastatic breast cancer, helping to improve the clinical outcome of breast cancer patients.
Ongoing cancer genome characterization studies continue to elucidate the spectrum of genomic abnormalities that drive many cancers, and in the clinical arena assessment of the driver genetic alterations in patients is playing an increasingly important diagnostic and/or prognostic role for many cancer types. However, the landscape of genomic abnormalities is still unknown for less common cancers, and the influence of specific genotypes on clinical behavior is often still unclear. To address some of these deficiencies, we developed Profile, a prospective cohort study to obtain genomic information on all patients at a large tertiary care medical center for cancer-related care. We enrolled patients with any cancer diagnosis, and, for each patient (unselected for cancer site or type) we applied mass spectrometric genotyping (OncoMap) of 471 common recurrent mutations in 41 cancer-related genes. We report the results of the first 5000 patients, of which 26% exhibited potentially actionable somatic mutations. These observations indicate the utility of genotyping in advancing the field of precision oncology.
Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.
Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.
Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It
has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively
monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept
study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines
tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets.
With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients
over the course of their disease.
Unlabelled:
Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function.
Significance:
Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer.
Purpose:
Increased circulating tumor cells (CTCs; five or more CTCs per 7.5 mL of whole blood) are associated with poor prognosis in metastatic breast cancer (MBC). A randomized trial of patients with persistent increase in CTCs tested whether changing chemotherapy after one cycle of first-line chemotherapy would improve the primary outcome of overall survival (OS).
Patients and methods:
Patients with MBC who did not have increased CTCs at baseline remained on initial therapy until progression (arm A). Patients with initially increased CTCs that decreased after 21 days of therapy remained on initial therapy (arm B). Patients with persistently increased CTCs after 21 days of therapy were randomly assigned to continue initial therapy (arm C1) or change to an alternative chemotherapy (arm C2).
Results:
Of 595 eligible and evaluable patients, 276 (46%) did not have increased CTCs (arm A). Of those with initially increased CTCs, 31 (10%) were not retested, 165 were assigned to arm B, and 123 were randomly assigned to arm C1 or C2. No difference in median OS was observed between arm C1 and C2 (10.7 and 12.5 months, respectively; P = .98). CTCs were strongly prognostic. Median OS for arms A, B, and C (C1 and C2 combined) were 35 months, 23 months, and 13 months, respectively (P < .001).
Conclusion:
This study confirms the prognostic significance of CTCs in patients with MBC receiving first-line chemotherapy. For patients with persistently increased CTCs after 21 days of first-line chemotherapy, early switching to an alternate cytotoxic therapy was not effective in prolonging OS. For this population, there is a need for more effective treatment than standard chemotherapy.
The promise of precision medicine will only be fully realized if the research community can adapt its clinical trials methodology to study molecularly characterized tumors instead of the traditional histologic classification. Such trials will depend on adequate tissue collection, availability of quality controlled, high throughput molecular assays, and the ability to screen large numbers of tumors to find those with the desired molecular alterations. The National Cancer Institute's (NCI) new National Clinical Trials Network (NCTN) is well positioned to conduct such trials. The NCTN has the ability to seamlessly perform ethics review, register patients, manage data, and deliver investigational drugs across its many sites including both in cities and rural communities, academic centers, and private practices. The initial set of trials will focus on different questions: (1) Exceptional Responders Initiative-why do a minority of patients with solid tumors or lymphoma respond very well to some drugs even if the majority do not?; (2) NCI MATCH trial-can molecular markers predict response to targeted therapies in patients with advanced cancer resistant to standard treatment?; (3) ALCHEMIST trial-will targeted epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibitors improve survival for adenocarcinoma of the lung in the adjuvant setting?; and (4) Lung Cancer Master Protocol trial for advanced squamous cell lung cancer-is there an advantage to developing drugs for small subsets of molecularly characterized tumors in a single, multiarm trial design? These studies will hopefully spawn a new era of treatment trials that will carefully select the tumors that may respond best to investigational therapy.
Background:
Oncogenic genetic alterations "drive" neoplastic cell proliferation. Small molecule inhibitors and antibodies are being developed that target an increasing number of these altered gene products. Next-generation sequencing (NGS) is a powerful tool to identify tumor-specific genetic changes. To determine the clinical impact of extensive genetic analysis, we reviewed our experience using a targeted NGS platform (FoundationOne) in advanced cancer patients.
Patients and methods:
We retrospectively assessed demographics, NGS results, and therapies received for patients undergoing targeted NGS (exonic sequencing of 236 genes and selective intronic sequencing from 19 genes) between April 2012 and August 2013. Coprimary endpoints were the percentage of patients with targeted therapy options uncovered by mutational profiling and the percentage who received genotype-directed therapy.
Results:
Samples from 103 patients were tested, most frequently breast carcinoma (26%), head and neck cancers (23%), and melanoma (10%). Most patients (83%) were found to harbor potentially actionable genetic alterations, involving cell-cycle regulation (44%), phosphatidylinositol 3-kinase-AKT (31%), and mitogen-activated protein kinase (19%) pathways. With median follow-up of 4.1 months, 21% received genotype-directed treatments, most in clinical trials (61%), leading to significant benefit in several cases. The most common reasons for not receiving genotype-directed therapy were selection of standard therapy (35%) and clinical deterioration (13%).
Conclusion:
Mutational profiling using a targeted NGS panel identified potentially actionable alterations in a majority of advanced cancer patients. The assay identified additional therapeutic options and facilitated clinical trial enrollment. As time progresses, NGS results will be used to guide therapy in an increasing proportion of patients.
Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.
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.
Non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib, but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor that has shown greater antitumor potency than crizotinib in preclinical studies.
In this phase 1 study, we administered oral ceritinib in doses of 50 to 750 mg once daily to patients with advanced cancers harboring genetic alterations in ALK. In an expansion phase of the study, patients received the maximum tolerated dose. Patients were assessed to determine the safety, pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies were performed before ceritinib treatment to identify resistance mutations in ALK in a group of patients with NSCLC who had had disease progression during treatment with crizotinib.
A total of 59 patients were enrolled in the dose-escalation phase. The maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic events included diarrhea, vomiting, dehydration, elevated aminotransferase levels, and hypophosphatemia. This phase was followed by an expansion phase, in which an additional 71 patients were treated, for a total of 130 patients overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib per day, the overall response rate was 58% (95% confidence interval [CI], 48 to 67). Among 80 patients who had received crizotinib previously, the response rate was 56% (95% CI, 45 to 67). Responses were observed in patients with various resistance mutations in ALK and in patients without detectable mutations. Among patients with NSCLC who received at least 400 mg of ceritinib per day, the median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5).
Ceritinib was highly active in patients with advanced, ALK-rearranged NSCLC, including those who had had disease progression during crizotinib treatment, regardless of the presence of resistance mutations in ALK. (Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number, NCT01283516.).
Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or “personalized”) medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR.
Significance: The study of exceptional responders represents a promising approach to better understanding the mechanisms that underlie sensitivity to targeted anticancer therapies. Here, we identify two activating mTOR mutations in a patient with exquisite sensitivity to everolimus and pazopanib, suggesting an approach to identifying patients who might benefit most from mTOR inhibitors. Cancer Discov; 4(5); 546–53. ©2014 AACR.
See related commentary by Rejto and Abraham, p. 513
This article is highlighted in the In This Issue feature, p. 495
Breast cancer is characterised by genomic alterations. We did a multicentre molecular screening study to identify abnormalities in individual patients with the aim of providing targeted therapy matched to individuals' genomic alterations.
From June 16, 2011, to July 30, 2012, we recruited patients who had breast cancer with a metastasis accessible for biopsy in 18 centres in France. Comparative genomic hybridisation (CGH) array and Sanger sequencing on PIK3CA (exon 10 and 21) and AKT1 (exon 4) were used to assess metastatic biopsy samples in five centres. Therapeutic targets were decided on the basis of identified genomic alterations. The primary objective was to include 30% of patients in clinical trials testing a targeted therapy and, therefore, the primary outcome was the proportion of patients to whom a targeted therapy could be offered. For the primary endpoint, the analyses were done on the overall population registered for the trial. This trial is registered with ClinicalTrials.gov, number NCT01414933.
423 patients were included, and biopsy samples were obtained from 407 (metastatic breast cancer was not found in four). CGH array and Sanger sequencing were feasible in 283 (67%) and 297 (70%) patients, respectively. A targetable genomic alteration was identified in 195 (46%) patients, most frequently in PIK3CA (74 [25%] of 297 identified genomic alterations), CCND1 (53 [19%]), and FGFR1 (36 [13%]). 117 (39%) of 297 patients with genomic tests available presented with rare genomic alterations (defined as occurring in less than 5% of the general population), including AKT1 mutations, and EGFR, MDM2, FGFR2, AKT2, IGF1R, and MET high-level amplifications. Therapy could be personalised in 55 (13%) of 423 patients. Of the 43 patients who were assessable and received targeted therapy, four (9%) had an objective response, and nine others (21%) had stable disease for more than 16 weeks. Serious (grade 3 or higher) adverse events related to biopsy were reported in four (1%) of enrolled patients, including pneumothorax (grade 3, one patient), pain (grade 3, one patient), haematoma (grade 3, one patient), and haemorrhagic shock (grade 3, one patient).
Personalisation of medicine for metastatic breast cancer is feasible, including for rare genomic alterations.
French National Cancer Institute, Breast Cancer Research Foundation, Odyssea, Operation Parrains Chercheurs.
Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.
We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations.
We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2-) and, as controls, 115 ER negative (ER-) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes.
Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naïve ER+ cancer or in any stage of ER- disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments.
In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer.
Unlabelled:
Neoadjuvant chemotherapy (NAC) induces a pathologic complete response (pCR) in approximately 30% of patients with triple-negative breast cancers (TNBC). In patients lacking a pCR, NAC selects a subpopulation of chemotherapy-resistant tumor cells. To understand the molecular underpinnings driving treatment-resistant TNBCs, we performed comprehensive molecular analyses on the residual disease of 74 clinically defined TNBCs after NAC, including next-generation sequencing (NGS) on 20 matched pretreatment biopsies. Combined NGS and digital RNA expression analysis identified diverse molecular lesions and pathway activation in drug-resistant tumor cells. Ninety percent of the tumors contained a genetic alteration potentially treatable with a currently available targeted therapy. Thus, profiling residual TNBCs after NAC identifies targetable molecular lesions in the chemotherapy-resistant component of the tumor, which may mirror micrometastases destined to recur clinically. These data can guide biomarker-driven adjuvant studies targeting these micrometastases to improve the outcome of patients with TNBC who do not respond completely to NAC.
Significance:
This study demonstrates the spectrum of genomic alterations present in residual TNBC after NAC. Because TNBCs that do not achieve a CR after NAC are likely to recur as metastatic disease at variable times after surgery, these alterations may guide the selection of targeted therapies immediately after mastectomy before these metastases become evident.
Resistance to endocrine therapy occurs in virtually all patients with estrogen receptor α (ERα)-positive metastatic breast cancer, and is attributed to various mechanisms including loss of ERα expression, altered activity of coregulators, and cross-talk between the ERα and growth factor signaling pathways. To our knowledge, acquired mutations of the ERα have not been described as mediating endocrine resistance. Samples of 13 patients with metastatic breast cancer were analyzed for mutations in cancer-related genes. In five patients who developed resistance to hormonal therapy, a mutation of A to G at position 1,613 of ERα, resulting in a substitution of aspartic acid at position 538 to glycine (D538G), was identified in liver metastases. Importantly, the mutation was not detected in the primary tumors obtained prior to endocrine treatment. Structural modeling indicated that D538G substitution leads to a conformational change in the ligand-binding domain, which mimics the conformation of activated ligand-bound receptor and alters binding of tamoxifen. Indeed, experiments in breast cancer cells indicated constitutive, ligand-independent transcriptional activity of the D538G receptor, and overexpression of it enhanced proliferation and conferred resistance to tamoxifen. These data indicate a novel mechanism of acquired endocrine resistance in breast cancer. Further studies are needed to assess the frequency of D538G-ERα among patients with breast cancer and explore ways to inhibit its activity and restore endocrine sensitivity. Cancer Res; 1-9. ©2013 AACR.
Seventy percent of breast cancers express estrogen receptor (ER), and most of these are sensitive to ER inhibition. However, many such tumors for unknown reasons become refractory to inhibition of estrogen action in the metastatic setting. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER-positive breast tumors and identified mutations in ESR1 affecting the ligand-binding domain (LBD) in 14 of 80 cases. These included highly recurrent mutations encoding p.Tyr537Ser, p.Tyr537Asn and p.Asp538Gly alterations. Molecular dynamics simulations suggest that the structures of the Tyr537Ser and Asp538Gly mutants involve hydrogen bonding of the mutant amino acids with Asp351, thus favoring the agonist conformation of the receptor. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD-mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may be of substantial therapeutic benefit.
Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.
There are ethical concerns regarding the performance of biopsies in patients for research purposes. We examined our single-institution experience regarding acceptance, safety, and success rate with research biopsies in patients with breast cancer. Among patients with data from paired samples, receptor status agreement between primary and metastatic samples was examined, either on first recurrence or after progression on one or more lines of therapy. An IRB-approved prospective study at the Dana-Farber Cancer Institute collects research biopsies as additional passes at the time of a clinical biopsy (AB, additional biopsy) or as a separate procedure for banking purposes (RPOB, research purposes only biopsy). Biopsies are not linked to a specific therapeutic or correlative trial. Grade 2-5 adverse events are prospectively collected. 151 patients were included in the analytic cohort (total procedures = 161); 80.8 % underwent AB, 17.2 % underwent RPOB, and 2.0 % underwent both AB and RPOB. Most patients were white (88.7 %) with a performance status of 0-1 (94.0 %). 96.0 % of patients underwent a biopsy in the setting of known or suspected metastatic disease. Receptor status between primary cancer and recurrent research biopsies differed in 43.2 % of patients with available data (18.8 % among patients who underwent the research biopsy before any systemic treatment, 48.1 % after treatment). Tissue was successfully collected in 92.3 % of patients undergoing AB and 100 % patients undergoing RPOB. Only three (2.0 %) patients had adverse events ≥grade-2: one grade-2 pain; one grade-2 pneumothorax; and one grade-3 pain. Our experience suggests research biopsies can be performed safely with a high rate of successful tissue collection. Consistent with previous reports we found a high rate of discordance between primary and metastatic samples, which was even higher among treated patients. This supports continued efforts to study tissue samples at multiple points in a patient's disease course.
To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.
All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, ‘kataegis’, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.
The evidence for T-cell–mediated regression of human cancers such as non–small-cell lung carcinoma, renal cell carcinoma, and—in particular—melanoma after immunotherapy is strong. Anti-CTLA4 (ipilimumab) treatment has been approved for treatment of meta-static melanoma,1 and antibody-mediated blockade of PD-1, a second inhibitory receptor on T cells, has shown highly encouraging results in early clinical trials.2,3 Although the clinical activity of these treatments is apparent, it is still unknown which T-cell reactivities are involved in immunotherapy-induced cancer regression.4 T-cell reactivity against nonmutated tumor-associated self-antigens has been analyzed in patients treated with ipilimumab or with autologous tumor-infiltrating T cells, but the magnitude of the T-cell responses observed has been relatively modest.5,6 In part on the basis of such data, recognition of patient-specific mutant epitopes (hereafter referred to as neoantigens) has been suggested to be a potentially important component.7 A potential involvement of mutated epitopes in T-cell control would also fit well with the observation that the mutation load in sun-exposed melanomas is particularly high.8-10
Intriguingly, on the basis of animal model data, it has recently been suggested that (therapy-induced) analysis of T-cell reactivity against patient-specific neoantigens may be feasible through exploitation of cancer genome data.11,12 However, human data have thus far been lacking. Here we report a case of a patient with stage IV melanoma who exhibited a clinical response to ipilimumab treatment. Cancer exome–guided analysis of T-cell reactivity in this patient revealed reactivity against two neoantigens, including a dominant T-cell response against a mutant epitope of the ATR (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exome–guided analysis to dissect the effects of melanoma immunotherapy.
To the Editor: Dawson et al. (March 28 issue)(1) suggest that the detection of circulating tumor DNA in 58% of patients with metastatic breast cancer can be used as an effective indicator of tumor load during treatment with standard systemic therapies. The study does not address the clinical utility of circulating tumor DNA. Moreover, the authors claim that circulating tumor DNA represents a more effective monitoring tool than the enumeration of circulating tumor cells. This statement is incorrect, considering that the enumeration of circulating tumor cells proved the ability to predict prognosis and monitor treatment efficacy in all patients with . . .
The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA--RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.
Background: Although the spectrum of genomic alterations in primary, treatment-naïve breast tumors has been described, the genomic landscape of HER2+ MBC remains underexplored. Furthermore, tumor genomic alterations that arise after progression on anti-HER2 therapy are largely unknown.
Methods: We prospectively collected metastatic tumor biopsies from patients (pts) enrolled on TBCRC003 (NCT00470704), a phase II study evaluating the combination of lapatinib (L) and T in pts with HER2+ MBC who had varying degrees of prior T exposure. We performed WES on baseline metastatic biopsies and normal DNA from 57 pts. In 36 pts, we also performed WES on pre-treatment primary tumors. Tumors were analyzed for point mutations, insertions/deletions, and copy number alterations.
Results: Total accrual was 116 pts. 87 pts were registered in one of two efficacy cohorts: Cohort 1 included pts w no prior T for MBC. Pts with prior adjuvant T were included if the interval from last T to 1st recurrence > 12 months. Cohort 2 included pts with 1-2 prior lines of T for MBC or recurrence within 12 months of adjuvant T. An additional 29 pts were enrolled in a biomarker cohort (Cohort 3). Per-protocol efficacy analyses for 85 pts deemed evaluable are shown below:
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As we previously reported (Wagle et al, ASCO 2014), across 57 metastatic tumors, significant recurrently mutated genes were TP53 (n=30; 53%) and PIK3CA (n=19; 33%). The frequency of mutant TP53 and PIK3CA was not significantly different from 119 primary, treatment-naïve HER2+ tumors sequenced in the TCGA study (50%, p=0.8 and 27%, p=0.5, respectively). Recurrent copy number alterations were also similar to TCGA data.
Comparing the 38 pts who received any prior T with the 19 pts who did not, there was no significant difference in the incidence of mutant TP53 (53% vs 53%, p=1.0) and PIK3CA (37% vs 26%, p=0.6).
We identified mutations in the HER2 kinase domain in 4/38 pts who received prior T (11%), as compared to 0/19 T-naïve pts. HER2 kinase domain mutations have been identified in ∼2% of HER2-negative cancers but <1% of primary HER2+ cancers. 3 of the mutations were L755S, which has been shown to be resistant to L and sensitive to irreversible HER2 inhibitors. The 4th mutation was D742N, a novel kinase domain mutation. None of the 4 pts with HER2 kinase domain mutations had an objective response, though 1 pt had stable disease for 29 weeks.
An analysis comparing paired archival primary tumors and baseline metastatic biopsies from 36 pts to identify genomic alterations acquired or enriched in the metastatic tumors will be presented.
Conclusions: We present an analysis of the genomic landscape of HER2+ MBC, including comparisons between matched primary tumors and metastatic biopsies. Somatic HER2 kinase mutations in pts with HER2+ MBC treated with prior T suggests that these mutations may be involved in resistance to T, and may predict poor response to additional anti-HER2 therapy with combined L and T. Novel therapeutic approaches may be required for these pts.
Citation Format: Nikhil Wagle, Nancy U Lin, Andrea L Richardson, Ignaty Leshchiner, Ingrid A Mayer, Andres Forero-Torres, Timothy J Hobday, Elizabeth C Dees, Rita Nanda, Mothaffar F Rimawi, Hao Guo, William T Barry, Ron Bose, Wei Shen, Antonio C Wolff, Stacey B Gabriel, Levi A Garraway, Eric P Winer, Ian E Krop. Whole exome sequencing (WES) of HER2+ metastatic breast cancer (MBC) from patients with or without prior trastuzumab (T): A correlative analysis of TBCRC003 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-5.
Triple negative breast cancer (TNBC) represents a particularly aggressive and difficult to treat form of breast cancer. No specific genetic alterations have been described as characteristic of the disease, with the exception of association with BRCA1/2, EGFR, and KRAS mutations. In this study, we sought to define clinically actionable mutations in untreated metastatic tumors as well as compare the mutational status of metastatic samples with germ-line and primary tumors using whole exome sequencing.
We prospectively enrolled 38 patients with newly diagnosed metastatic TNBC and collected matched specimens of germ-line DNA, primary tumor and metastatic tumor. Median DFI from time of initial primary diagnosis to recurrence was 18 months (IQR = 1-24 months) and 9 patients presented with de novo metastatic disease. 34/38 patients went on to receive first-line treatment with nab-paclitaxel, carboplatin, and bevacizumab and ORR/PFS/OS are available.
Sites of TNBC metastatic tissue (n = 31) included: liver (10), chest wall (13), non-regional lymph nodes (4), and lung (4). 7 patients had inadequate metastatic tumor for sequencing. We performed whole-exome sequencing for all samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 10 GB of sequencing data using high throughput sequencing on the Illumina platform.
We observed striking genetic heterogeneity among the metastatic and primary tumors. There was no single driver mutation that was common to the metastatic tumors indicating the diverse genetic pathways that contribute to metastasis. Early analysis suggests that mutations in APC and MTOR occur more frequently in metastatic tumors than in primary tumors. Nonsense mutations of ER were detected in both primary and metastatic tumors but not in germ-line DNA. EGFR and HER2 mutations were not found in any of the primary or metastatic TNBC samples.
This data provides the most comprehensive genetic portrait of metastatic and primary TNBC to date, and represents a significant first step in identifying the genetic causes of the disease, drivers of recurrence, and potential therapeutic targets. Full results, including the primary versus metastatic tumor mutational analysis will be presented.
This study was funded by a Susan G. Komen Grant SAC 100001.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S4-03.
Background: LCIS has traditionally been recognized as a marker of increased risk for the subsequent development of breast cancer, of either the lobular or ductal phenotype, yet due to the incidental nature of LCIS little is known about its underlying biology. Here we describe the first report of novel mutations in LCIS using targeted exome sequencing of fresh frozen tissue samples.
Methods: Fresh frozen tissue samples from patients with a prior history of LCIS undergoing therapeutic or risk-reducing mastectomy from 2003–08 were harvested and systematically reviewed to identify LCIS. Cells from individual LCIS lesions +/− associated cancers were collected by laser capture microdissection. For the purposes of this study, germline DNA (blood) and DNA from 12 unique LCIS lesions were subject to targeted parallel sequencing of exons corresponding to 230 cancer genes using the Illumina HiSeq 2000 platform. DNA from an associated ductal carcinoma in situ (DCIS) and/or an invasive ductal (IDC) or lobular (ILC) lesion was also available for 7 of these cases resulting in 41 samples from 12 pts for mutational analysis. Normalized (RMA) Affymetrix U133A gene expression data were also available.
Results: DNA profiling reliably identified 7 somatic mutations in 5/12 LCIS samples (41.7%). Of these, 4/7 mutations were base substitutions (missense mutations); and the others included: 1 deletion; 1 silent and 1 splice-site mutation. Mutations in LCIS were identified in 5/230 cancer genes analyzed, including: PIK3CA, CDH1, NOTCH4, PREX2 and ARAF. PIK3CA and CDH1 mutations were each identified in two samples, representing 4/7 (57.1%) mutations. Specific mutations found in LCIS and their frequencies are listed (table). Among 3 LCIS-ILC pairs, one shared the G914R mutation in PIK3CA, and 1/3 LCIS-IDC pairs exhibited an identical point mutation (R373W) in the NOTCH4 gene. No shared mutations were observed in 3 LCIS-DCIS pairs. Both CDH1 mutated cases were associated with decreased e-cadherin mRNA levels when compared to non-mutated cases (mean 9.88 vs 11.01), as was the NOTCH4 mutation (mean 6.02 vs 6.47). Mutations in ARAF and PREX2 were associated with increased mRNA levels, mean 7.07 vs 6.52 and 4.82 vs 4.22, respectively. The hotspot PIK3CA mutation (E545K) was also associated with increased gene expression (mean 5.15 vs 4.64) whereas the G914R mutant was associated with decreased expression (mean 4.13 vs 4.64).
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Conclusions: This study represents the first targeted exon sequencing analysis of fresh frozen LCIS. Although LCIS has been regarded as a rather genetically stable lesion, somatic mutations were detected in 41.7% of lesions in this small cohort. While CDH1 mutations are expected in lobular neoplasia, this is the first report of mutations in ARAF, NOTCH4, PIK3CA and PREX2. Given the shared relevance of PIK3CA and PREX2 in the PI3K/AKT pathway, these findings suggest novel mechanisms for new chemoprevention strategies among women with LCIS.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-02.
Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.
Background:
Combined BRAF and MEK inhibition, as compared with BRAF inhibition alone, delays the emergence of resistance and reduces toxic effects in patients who have melanoma with BRAF V600E or V600K mutations.
Methods:
In this phase 3 trial, we randomly assigned 423 previously untreated patients who had unresectable stage IIIC or stage IV melanoma with a BRAF V600E or V600K mutation to receive a combination of dabrafenib (150 mg orally twice daily) and trametinib (2 mg orally once daily) or dabrafenib and placebo. The primary end point was progression-free survival. Secondary end points included overall survival, response rate, response duration, and safety. A preplanned interim overall survival analysis was conducted.
Results:
The median progression-free survival was 9.3 months in the dabrafenib-trametinib group and 8.8 months in the dabrafenib-only group (hazard ratio for progression or death in the dabrafenib-trametinib group, 0.75; 95% confidence interval [CI], 0.57 to 0.99; P=0.03). The overall response rate was 67% in the dabrafenib-trametinib group and 51% in the dabrafenib-only group (P=0.002). At 6 months, the interim overall survival rate was 93% with dabrafenib-trametinib and 85% with dabrafenib alone (hazard ratio for death, 0.63; 95% CI, 0.42 to 0.94; P=0.02). However, a specified efficacy-stopping boundary (two-sided P=0.00028) was not crossed. Rates of adverse events were similar in the two groups, although more dose modifications occurred in the dabrafenib-trametinib group. The rate of cutaneous squamous-cell carcinoma was lower in the dabrafenib-trametinib group than in the dabrafenib-only group (2% vs. 9%), whereas pyrexia occurred in more patients (51% vs. 28%) and was more often severe (grade 3, 6% vs. 2%) in the dabrafenib-trametinib group.
Conclusions:
A combination of dabrafenib and trametinib, as compared with dabrafenib alone, improved the rate of progression-free survival in previously untreated patients who had metastatic melanoma with BRAF V600E or V600K mutations. (Funded by GlaxoSmithKline; Clinical Trials.gov number, NCT01584648.).
Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.
Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication.
To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings.
An exploratory study of 12 adult participants recruited at Stanford University Medical Center who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings.
Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up.
Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross κ, 0.52; 95% CI, 0.40-0.64), and reclassified 69% of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss κ, 0.24; P < 001).
In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine.
Next generation sequencing technology is increasingly utilized in oncology with the goal of targeting therapeutics to improve response and reduce side effects. Interpretation of tumor mutations requires sequencing of paired germline DNA, raising questions about incidental germline findings. We describe our experiences as part of a research team implementing a protocol for whole genome sequencing (WGS) of tumors and paired germline DNA known as the Michigan Oncology Sequencing project (MI-ONCOSEQ) that includes options for receiving incidental germline findings. Genetic counselors (GCs) discuss options for return of results with patients during the informed consent process and document family histories. GCs also review germline findings and actively participate in the multi-disciplinary Precision Medicine Tumor Board (PMTB), providing clinical context for interpretation of germline results and making recommendations about disclosure of germline findings. GCs have encountered ethical and counseling challenges with participants, described here. Although GCs have not been traditionally involved in molecular testing of tumors, our experiences with MI-ONCOSEQ demonstrate that GCs have important applicable skills to contribute to multi-disciplinary care teams implementing precision oncology. Broader use of WGS in oncology treatment decision making and American College of Medical Genetics and Genomics (ACMG) recommendations for active interrogation of germline tissue in tumor-normal dyads suggests that GCs will have future opportunities in this area outside of research settings.
The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in similar to 40% of genes, with the landscape dominated by cis-and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA-RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the 'CNA-devoid' subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP–ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2Q60P). MEK2Q60P conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal–regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.
Significance: This study represents an initial clinical genomic study of acquired resistance to combined RAF/MEK inhibition in BRAF-mutant melanoma, using WES and RNA-seq. The presence of diverse resistance mechanisms suggests that serial biopsies and genomic/molecular profiling at the time of resistance may ultimately improve the care of patients with resistant BRAF-mutant melanoma by specifying tailored targeted combinations to overcome specific resistance mechanisms. Cancer Discov; 4(1); 61–8. ©2013 AACR.
See related commentary by Solit and Rosen, p. 27
This article is highlighted in the In This Issue feature, p. 1
A 53-year-old woman was referred to our institution fortreatment recommendations for her advanced inflammatorybreast carcinoma (IBC) that continued to progress locally despiteoptimal HER2-targeted therapy. She initially presented 2 yearsearlier with clinical signs suggestive of a right IBC. She underwentadiagnosticcoreneedlebiopsy,which demonstrated an invasiveductal carcinoma, Grade 3, with dermolymphatic emboli. Theprimary tumor tested negative for the expression of estrogenreceptor (ER) and progesterone receptor (PR) using immuno-histochemistry (IHC) and was HER2 3þ using IHC. At the timeof initial diagnosis the tumor had metastasized to multiple bonesand the liver, and the patient was staged as T4dN1M1 (liver andbone) stage IV.The patient began treatment with trastuzumab (Herceptin)combined with docetaxel (Taxotere) and carboplatin (Paraplatin)and achieved an excellent clinical and radiological response. Eightmonths later, the patient developed expressive aphasia, and mag-netic resonance imaging (MRI) and computed tomography imagingconfirmed a single metastatic lesion in the left frontoparietal region.Subsequent craniotomy revealed a 2.4-cm ER-negative/PR-negative/HER2 3þ metastasis. A month later, a MRI scanrevealed a second 0.5-cm brain metastasis that was subsequentlytreated with gamma knife irradiation.Considering the persistence of systemic disease in other sites,chemotherapy with capecitabine (Xeloda) and laptinib (Tykerb) wasinitiated.Threemonthslater,thepatientagainrequiredtreatmentwithgamma knife irradiation for 2 additional cerebral metastases. Shecontinued the treatment with apparent clinical bene fit, but 4 monthslater,imagingforrestagingshowedprogressivein filtrationintothechestwall and increased metabolic activity in the local lymph nodes. Tras-tuzumab was added to the current treatment regimen. Despite a
To the Editor: Dawson et al. (March 28 issue)(1) suggest that the detection of circulating tumor DNA in 58% of patients with metastatic breast cancer can be used as an effective indicator of tumor load during treatment with standard systemic therapies. The study does not address the clinical utility of circulating tumor DNA. Moreover, the authors claim that circulating tumor DNA represents a more effective monitoring tool than the enumeration of circulating tumor cells. This statement is incorrect, considering that the enumeration of circulating tumor cells proved the ability to predict prognosis and monitor treatment efficacy in all patients with . . .