Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle

Mattos-Arruda, Leticia De; Weigelt, Britta; Cortés, Javier; Won, Helen; Ng, Charlotte K.Y.; Nuciforo, Paolo; Bidard, François-Clément; Aura, Claudia; Saura, Cristina; Peg, Vicente; Piscuoglio, Salvatore; Oliveira, Mafalda; Smolders, Y.; Patel, Purvi; Norton, Larry; Tabernero, Josep; Berger, Michael F.; Seoane, Joan; Reis‐Filho, Jorge S.

doi:10.1093/annonc/mdu239

Cited by 326 publications

(263 citation statements)

References 31 publications

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“…The very low allele frequency suggests that in some primary tumors ESR1 mutations pre-exist as rare clones, which are then selected for during metastatic progression. This is consistent with a previous study from a single patient, which used deep-targeted MPS and identified an ESR1 mutation (E380Q) at 2% allele frequency in primary disease and 68% in synchronous liver metastasis (25, 28). Detection of rare ESR1 mutations in primary tumors (0–7%) may be clinically relevant for predicting resistance to hormone therapy; however, additional studies using sensitive detection technologies are necessary to develop this area of investigation.…”

Section: Discussionsupporting

confidence: 93%

“…The feasibility of using cfDNA to noninvasively identify molecular alterations within metastatic tumors has been shown in several studies (24–26) and preliminary data suggest that cfDNA can be used to monitor breast cancer burden and treatment response (27). A recent proof-of-principle study detected an ESR1 mutation (E380Q) in cfDNA from a single patient with advanced hormone refractory breast cancer (25, 28). However, the detection of rare mutations has been challenged by several limiting factors, including low cfDNA yields and low tumor cellularity in metastatic lesions.…”

Section: Introductionmentioning

confidence: 99%

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Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Wang

Bahreini

Gyanchandani

et al. 2016

Clinical Cancer Research

167

168

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Purpose Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell free DNA (cfDNA). Patients and Methods Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n=43), bone (n=12) and brain metastases (n=38), and cfDNA (n=29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results ESR1 mutations were detected for D538G (n=13), Y537S (n=3) and Y537C (n=1), and not for K303R, S463P or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3 to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n=5), mutations were detected in both (n=3) or in cfDNA only (n=2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1 mutant clones are enriched by endocrine therapy. Further studies should address if sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Wang

Bahreini

Gyanchandani

et al. 2016

Clinical Cancer Research

167

168

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“…Recent studies demonstrate that ctDNA can detect intratumor and multitumor heterogeneity and detect complex patterns of treatment resistance. 52–55 With the emergence of new sequencing modifications that improve sensitivity and decrease sequencing errors, we believe that ctDNA profiling by next-generation sequencing approaches will improve our understanding of tumor heterogeneity and patterns of somatic evolution in pediatric solid tumors. 12,16 In addition, the assays described in this study could facilitate broader profiling of ctDNA, such as deep whole-exome sequencing, by providing a mechanism to screen samples for the presence of sufficiently abundant ctDNA, allowing selection of samples most likely to yield informative data.…”

Section: Discussionmentioning

confidence: 99%

Detection of Somatic Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in Children With Solid Tumors

Klega

Imamovic-Tuco

et al. 2018

JCO Precision Oncology

145

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Objective Liquid biopsies are being rapidly used in adult cancers as new biomarkers of disease. Circulating tumor DNA (ctDNA) levels have been reported to be proportional to disease burden, correlate with treatment response, and predict relapse. However, little is known about how frequently ctDNA is detectable in pediatric patients with solid tumors. Therefore, we developed a next-generation sequencing approach to detect and quantify ctDNA in the blood of patients with the most common pediatric solid tumors. Methods Detection of ctDNA requires assays sensitive to somatic events typically observed in the cancer type being studied. In pediatric solid tumors, structural variants are more common than recurrent point mutations. We adapted an ultralow passage whole-genome sequencing approach to capture copy number variants and a hybrid capture sequencing assay to detect translocations in liquid biopsy samples from pediatric patients. Results Copy number changes seen by ultralow passage whole-genome sequencing enabled detection of ctDNA in patients with osteosarcoma, neuroblastoma, alveolar rhabdomyosarcoma, and Wilms tumor. In Ewing sarcoma, detection of the EWSR1 translocation was a more sensitive approach. For patients with samples collected at multiple time points, changes in ctDNA levels corresponded to treatment response. We also found that disease-specific genomic biomarkers of prognosis were detectable in ctDNA. Conclusion This study demonstrates that liquid biopsy approaches that detect somatic structural variants are well suited to pediatric solid tumors. We show that children with the most common solid tumor malignancies have detectable levels of ctDNA, which may be used to track disease response and identify genomic subclassifiers of disease. Efforts to profile larger collections of clinically annotated specimens are under way to validate the clinical use of these assays.

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“…Mutations in the derivative cell lines that were not detected in the BT474/ATCC parental cell line (BT474/ATCC-P) were defined using MuTect (28) for the single nucleotide variants, and a combination of Strelka and Varscan2 (29, 30) for insertions and deletions, with the BT474/ATCC-P line as the reference. SNVs and indels with mutant allelic fraction of less than 1% and/or supported by fewer than 5 reads were disregarded (31). Variants found with >5% global minor allele frequency in dbSNP (Build 137) or that were covered by <10 reads in the tumor or <5 reads in the BT474/ATCC-P cell line were disregarded.…”

Section: Methodsmentioning

confidence: 99%

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Angelis

Burke

et al. 2017

Clinical Cancer Research

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Purpose Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the L-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared to parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired L resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2 irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusion HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to L-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors.

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Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle

Abstract: www.clinicaltrials.gov, NCT01090960.

Cited by 326 publications

References 31 publications

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Detection of Somatic Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in Children With Solid Tumors

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

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