Sequencing Of Circulating Tumor DNA Research Articles

Comprehensive Analysis of TP53 Mutation Characteristics and Identification of Patients with Inferior Prognosis and Enhanced Immune Escape in Diffuse Large B Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphatic malignancy from both the clinical and molecular standpoints. TP53 mutations have been observed in DLBCL with high frequency mutations, which play a crucial role in lymphomagenesis. Considering the complex pathophysiological mechanisms involved in DLBCL, we herein assume that the interaction of TP53 with other genetic variants together promote the development of DLBCL. We also explore that these genetic interactions shape the discrepant immune landscape in DLBCL.Methods: We performed a comprehensive analysis of TP53 mutations in the DNA-binding domain (DBD) region. Genome alterations that were mutually exclusive or co-occurring with TP53 mutations were identified, and their potential value in prognosis and possible relationships with tumor microenvironment (TME) were further examined using targeted next-generation sequencing (n=176), transcriptome sequencing (n=152) and circulating tumor DNA sequencing (n=38).Results: TP53 was frequently mutated in DLBCL with a rate of 30% (53 of 176). The occurrence of TP53 mutations was skewed toward the early stages of DLBCL pathogenesis. Among these variants of TP53, 74% were missense mutations, and the remaining were inactivating frameshift indels, nonsense mutations, coding sequencing indels and splicing mutations. Importantly, most mutations (87.5%) occurred in exons 5-8, which encoded the DNA-binding domain (DBD) region of TP53. Codons 175, 273, and 248 of the p53 protein had the highest mutation frequency, which are also the hot spots of TP53 mutation found in most human cancers. However, TP53 alone is insufficient to effectively differentiate the risk of DLBCL, even when only considering mutations in the DBD region. CD58 mutations, which are mutually exclusive from TP53 mutations, in combination with TP53 mutations, could significantly differentiate the prognosis of DLBCL. The survival of patients with either one of the mutually exclusive mutation patterns, namely, TP53MUT&CD58WT or TP53WT&CD58MUT, was inferior to those harboring both wild-type TP53 and CD58. Notably, patients with TP53WT&CD58MUT mutation pattern had the worst outcome and were characterized by a higher overall tumor mutation burden (TMB) and immune score. An enhanced immune escape feature, including the abundant infiltration of inflammatory cells and upregulation of inhibitory immunomodulatory molecules such as PD-1, TIM3, LAG3, KLRC1, KLRC3, KLRD1, IDO1, IDO2 and TDO2, was observed in these patients with TP53WT&CD58MUT mutation pattern. A larger number of terms related to cytokine and chemokine and inflammatory pathways were enriched in the TP53WT&CD58MUT group, including cytokine and chemokine production, binding and activity, and interferon-γ pathway. Additionally, GSEA demonstrated that interferon-α and -γ responses and IL-6/JAK/STAT3 signaling enriched in the TP53WT&CD58MUT group. These patients with TP53WT&CD58MUT mutation pattern represent the candidate populations for immune therapy.Conclusions: Our findings indicated that the mutation patterns of TP53 and CD58 accurately stratified patients with DLBCL to permit the optional immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Feasibility of Using Biomarkers Derived from Circulating Tumor DNA Sequencing as Predictive Classifiers in Patients with Small-Cell Lung Cancer.

PurposeThis study aimed to investigate the feasibility of biomarkers based on dynamic circulating tumor DNA (ctDNA) to classify small cell lung cancer (SCLC) into different subtypes.Materials and MethodsTumor and longitudinal plasma ctDNA samples were analyzed by next-generation sequencing of 1,021 genes. PyClone was used to infer the molecular tumor burden index (mTBI). Pre-treatment tumor tissues (T1) and serial plasma samples were collected (pre-treatment [B1], after two [B2], six [B3] cycles of chemotherapy and at progression [B4]).ResultsOverall concordance between T1 and B1 sequencing (n=30) was 66.5%, and 89.5% in the gene of RB1. A classification method was designed according to the changes of RB1 mutation, named as subtype I (both positive at B1 and B2), subtype II (positive at B1 but negative at B2), and subtype III (both negative at B1 and B2). The median progressive-free survival for subtype I patients (4.5 months [95% confidence interval (CI), 2.6 to 5.8]) was inferior to subtype II (not reached, p < 0.001) and subtype III (10.8 months [95% CI, 6.0 to 14.4], p=0.002). The median overall survival for subtype I patients (16.3 months [95% CI, 5.3 to 22.9]) was inferior to subtype II (not reached, p=0.01) and subtype III (not reached, p=0.02). Patients with a mTBI dropped to zero at B2 had longer median overall survival (not reached vs. 19.5 months, p=0.01). The changes of mTBI from B4 to B1 were sensitive to predict new metastases, with a sensitivity of 100% and a specificity of 85.7%.ConclusionMonitoring ctDNA based RB1 mutation and mTBI provided a feasible tool to predict the prognosis of SCLC.

Circulating tumor DNA sequencing in colorectal cancer patients treated with first-line chemotherapy with anti-EGFR

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (< 1%) at the first evaluation showed significantly better PFS (p < 0.001), and the average VAF change further discriminated the PFS in the patients in partial response (p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.

The Feasibility of Using Biomarkers Derived from Circulating Tumor DNA Sequencing as Predictive Classifiers in Patients with Small-Cell Lung Cancer

Background: Exploring predictive biomarkers to individualize patients with small cell lung cancer (SCLC) remains challenging in clinical practice, and current imaging techniques cannot fully meet the clinical needs due to their inherent limitations. Methods: A total of 127 longitudinal biological DNA samples from 35 SCLC patients treated with first-line standard therapy were analyzed by capture-based targeted (average coverage for tissue and circulating tumor DNA (ctDNA) samples were 841× and 2670.5×, respectively) genome next-generation sequencing, which was designed to cover coding sequencing or hot exons of 1,021 genes frequently mutated in solid tumors. PyClone was used to infer the molecular tumor burden index (mTBI). Pre-treatment tumor tissues [T1] and serial plasma samples were collected (pre-treatment [B1], after two [B2], six [B3] cycles of chemotherapy and at progression [B4]). Findings: In total, 38 SCLC patients were enrolled with 35 patients included in the analysis set. The overall concordance rate between tissue and plasma sequencing in 30 patients with matched T1 and B1 samples was 66.5%. The median progression-free survival (PFS) was 9.8 months (95% confidence interval [CI]: 6.3-13.3). Patients with persisted RB1 and/or TP53 mutations at B2 demonstrated inferior PFS compared with others (5.2 months [95%CI: 3.8-6.6] vs. 13.1 months [95%CI: 9.8-16.4], p=0.06). The median PFS for patients with persisted RB1 mutations at B2 was significantly worse than the other two groups (4.2 months [95%CI: 2.8-5.6] vs. not reached, p<0.0001 and vs. 10.2 months, [95%CI: 6.3-14.1], p=0.001). The performance of mTBI monitoring was highly consistent with tumor imaging, and earlier mTBI elevation was observed in 64.7% of patients with B3 samples. In addition, the dynamic changes of mTBI from B4 to B1 were sensitive to predict new metastases, with sensitivity and specificity of 100% and 85.7% (95%CI: 50.8-120.7), respectively. Interpretation: Monitoring ctDNA based RB1 mutations and mTBI provided a feasible tool to predict prognosis of first-line therapy in SCLC, which guaranteed the development of prospective stratification in future trials. Trial Registration: This study was registered at the Chinese Clinical Trial Registry as ChiCTR1900023956. Funding: This study was supported by the National Key Research and Development Project (2019YFC1315704, to ZW), CAMS Innovation Fund for Medical Sciences (2017-I2M-1-005, to ZW), the National Natural Sciences Foundation (81871889, 82072586 to ZW) and Beijing Natural Science Foundation (7212084 to ZW). Declaration of Interest: The authors declare no potential competing interests. Ethical Approval: All procedures were conducted in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China; approved No. 18- 151/1729).

Plasma Circulating Tumor DNA Sequencing Predicts Minimal Residual Disease in Resectable Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is lethal as tumors are rarely detected at an early stage and have a high recurrence rate. There are no particularly useful biomarkers for the prognostic prediction of ESCC. Circulating tumor DNA (ctDNA) is becoming an important biomarker for non-invasive diagnosis and monitoring tumor prognosis. Here, we aimed to analyze variations in plasma cell-free DNA (cfDNA) amount to search for minimal residual disease (MRD). Plasma and white blood cells (WBCs) of 60 patients were collected before tumor resection and a week after surgery. Tumor specimens were also collected as formalin-fixed paraffin-embedded (FFPE) samples. All samples were extracted to analyze the genetic alterations of 61 genes using capture-based next-generation sequencing (NGS). Tumor variants were detected in 38 patients with ESCC, and the two driver genes with the highest mutation frequency were TP53 and PIK3CA. Of the pre-surgical plasma cfDNA samples, 73.7% of identified variants matched the tissue. In patients who did not receive adjuvant therapy after surgery, postoperative cfDNA-positive patients had shorter overall survival (hazard ratios (HR), 25.8; 95% CI, 2.7–242.6; P = 0.004) and were more likely to relapse than postoperative cfDNA-negative patients (HR, 184.6; 95% CI, 3.6–9576.9; P = 0.01). Detection of ctDNA after surgical tumor excision is associated with tumor relapse and disease-specific survival, and can be used as a prognostic biomarker for MRD detection in ESCC.

Genomic profile of advanced breast cancer in circulating tumour DNA

The genomics of advanced breast cancer (ABC) has been described through tumour tissue biopsy sequencing, although these approaches are limited by geographical and temporal heterogeneity. Here we use plasma circulating tumour DNA sequencing to interrogate the genomic profile of ABC in 800 patients in the plasmaMATCH trial. We demonstrate diverse subclonal resistance mutations, including enrichment of HER2 mutations in HER2 positive disease, co-occurring ESR1 and MAP kinase pathway mutations in HR + HER2− disease that associate with poor overall survival (p = 0.0092), and multiple PIK3CA mutations in HR + disease that associate with short progression free survival on fulvestrant (p = 0.0036). The fraction of cancer with a mutation, the clonal dominance of a mutation, varied between genes, and within hotspot mutations of ESR1 and PIK3CA. In ER-positive breast cancer subclonal mutations were enriched in an APOBEC mutational signature, with second hit PIK3CA mutations acquired subclonally and at sites characteristic of APOBEC mutagenesis. This study utilises circulating tumour DNA analysis in a large clinical trial to demonstrate the subclonal diversification of pre-treated advanced breast cancer, identifying distinct mutational processes in advanced ER-positive breast cancer, and novel therapeutic opportunities.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology.

Decoding the Evolutionary Response to Ensartinib in Patients With ALK-Positive NSCLC by Dynamic Circulating Tumor DNA Sequencing

IntroductionBy implementing dynamic circulating tumor DNA (ctDNA) analysis, we explored the impact of TP53 mutations on tumor evolution and resistance mechanisms to ensartinib in patients with ALK-positive NSCLC. MethodsIn a multicenter phase 2 trial, patients with ALK-positive NSCLC who progressed on crizotinib were treated with ensartinib. Blood samples for ctDNA analysis were collected at baseline, cycle 3 day 1, and progression disease (PD) and analyzed with a 212-gene panel. ResultsA total of 440 samples were collected from 168 patients. Baseline TP53 mutations (20.2%) significantly correlated with inferior progression-free survival (4.2 mo versus 11.7 mo, p < 0.0001). Patients with TP53 mutations had higher mutation load than those without TP53 mutations at baseline (13.79 ± 3.72 versus 4.67 ± 0.39, p < 0.001). Although there was no significant difference in mutation load between these groups at cycle 3 day 1 (5.89 ± 2.25 versus 3.72 ± 0.62, p = 0.425), patients with mutated TP53 developed more mutations at PD (7.07 ± 1.25 versus 3.20 ± 0.33, p = 0.003). Frequency and abundance of secondary ALK mutations G1269A, G1202R, and E1210K increased markedly at PD than baseline. In patients without secondary ALK mutations, we identified ALK-independent resistance mechanisms including bypass signaling activation, downstream effector protein reactivation, epithelial-mesenchymal transformation, and epigenetic dysregulation. ConclusionsOur study highlighted the advantage of ctDNA analysis for monitoring tumor evolution. TP53 mutations promoted genetic evolution and accelerated occurrence of resistance. We also unveiled ALK-dependent resistance mechanisms, mainly by G1269A, G1202R, and E1210K mutations, and ALK-independent resistance mechanisms to ensartinib.

A Targeted Gene Panel for Circulating Tumor DNA Sequencing in Neuroblastoma.

BackgroundLiquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants with low allele frequency. Most retrospective studies conducted in small cohorts of pediatric cancers have illustrated that the technology yield substantial potential in neuroblastoma.AimThe molecular landscape of neuroblastoma harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to characterize the mutational landscape of primary neuroblastoma using a custom gene panel for ctDNA targeted sequencing.MethodsTargeted next-generation sequencing (NGS) was performed on ctDNA of 11 patients with primary neuroblastoma stage 4. To avoid the detection of false variants, we used UMIs (unique molecular identifiers) for the library construction, increased the sequencing depth and developed ad hoc bioinformatic analyses including the hard filtering of the variant calls.ResultsWe identified 9/11 (81.8%) patients who carry at least one pathogenic variation. The most frequently mutated genes were KMT2C (five cases), NOTCH1/2 (four cases), CREBBP (three cases), ARID1A/B (three cases), ALK (two cases), FGFR1 (two cases), FAT4 (two cases) and CARD11 (two cases).ConclusionsWe developed a targeted NGS approach to identify tumor-specific alterations in ctDNA of neuroblastoma patients. Our results show the reliability of our approach to generate genomic information which can be integrated with clinical and pathological data at diagnosis.

Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy.

Simple SummaryRecent studies have shown the potential of next generation sequencing (NGS) for the identification of genetic variants in tumour DNA that has been released into the bloodstream (ctDNA). However, such variants are often rare in the sample and error correction is required to filter out false calls. In this study we used error corrected ctDNA-sequencing to identify genetic drivers of resistance in metastatic colorectal cancer (mCRC) patients that had become resistant to combined chemotherapy and EGFR-antibody treatment. Our data showed that ctDNA-seq could detect common and novel resistance mechanisms in cases of both primary and acquired resistance. ctDNA-seq could therefore facilitate patient stratification to novel therapies and avoid ineffective treatment with EGFR-antibodies. Furthermore, the data revealed a lack of detectable genetic resistance in a large fraction of the cancer cell population, indicating a need to investigate other, potentially non-genetic, resistance mechanisms.Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.

Considerations for the use of circulating tumor DNA sequencing as a screening tool in cancer predisposition syndromes.

Liquid biopsy, specifically circulating tumor DNA (ctDNA) detection, has started to revolutionize the clinical management of patients with cancer by surpassing many limitations of traditional tissue biopsies, particularly for serial testing. ctDNA sequencing has been successfully utilized for cancer detection, prognostication, and assessment of disease response and evolution. While the applications of ctDNA analysis are growing, the majority of studies to date have primarily evaluated its use as a tool for tracking aknown cancer,and in most cases at advanced stage. Herein, we discuss the potential application of ctDNA for surveillance and early cancer detection in patients with a cancer predisposition syndrome.

Detection of genomic alterations in breast cancer with circulating tumour DNA sequencing

Analysis of circulating cell-free DNA (cfDNA) has opened new opportunities for characterizing tumour mutational landscapes with many applications in genomic-driven oncology. We developed a customized targeted cfDNA sequencing approach for breast cancer (BC) using unique molecular identifiers (UMIs) for error correction. Our assay, spanning a 284.5 kb target region, is combined with a novel freely-licensed bioinformatics pipeline that provides detection of low-frequency variants, and reliable identification of copy number variations (CNVs) directly from plasma DNA. We first evaluated our pipeline on reference samples. Then in a cohort of 35 BC patients our approach detected actionable driver and clonal variants at low variant frequency levels in cfDNA that were concordant (77%) with sequencing of primary and/or metastatic solid tumour sites. We also detected ERRB2 gene CNVs used for HER2 subtype classification with 80% precision compared to immunohistochemistry. Further, we evaluated fragmentation profiles of cfDNA in BC and observed distinct differences compared to data from healthy individuals. Our results show that the developed assay addresses the majority of tumour associated aberrations directly from plasma DNA, and thus may be used to elucidate genomic alterations in liquid biopsy studies.

Circulating tumor DNA sequencing analysis of patients with metastatic renal cell carcinoma (mRCC).

e17100 Background: Despite extensive work to characterize genomic alterations (GAs) in metastatic renal cell carcinoma (mRCC), GAs are not currently used for treatment sequencing or selection. Circulating tumor DNA (ctDNA) in both blood and urine may elucidate potential mRCC biomarkers through a minimally invasive approach. We aimed to validate previous blood-based ctDNA studies (Pal et al Eur Urol 2017) and compare detection rates of clinically relevant GAs in plasma and urine. Methods: From a single institution, patients (pts) with mRCC were recruited to provide a plasma and urine specimen. ctDNA next-generation sequencing was performed using an investigational 120-gene panel, and we limited our assessment to 7 GAs with biological relevance in RCC ( VHL, MTOR, PIK3CA, TSC2, MET, AKT1, TSC1). Further, only pathogenic alterations previously recognized in RCC and cited in the Catalogue of Somatic Mutations in Cancer (COSMIC) database were considered. Clinicopathologic variables, treatment type and response to therapy were collected from an institutional mRCC patient database. Concordance analysis was performed at the gene level between blood and urine using previously published approaches (Chae et al Oncotarget 2016). Results: 50 pts (40M:10F) were enrolled with a median age of 65. 40 pts (80%) had clear cell histology, while the remainder were papillary (12%), chromophobe (4%) or other (4%). Pts received a median of 1 line of therapy (range, 0-7). GAs were identified in 45 pts (90%) in blood and in 45 pts (90%) in urine . Applying the above criteria, the most frequently observed GAs in blood specimens were TSC2 (9%), MTOR (4%), and VHL (2%). The most frequently observed GAs in urine were VHL (4%) and MTOR (2%). The rate of concordance between blood- and urine-detected GAs was 93%. Of pts bearing mTOR pathway alterations in blood or urine, 3 received everolimus-based therapy. All 3 pts remain on therapy at 10.4, 13.8 and 14.0 months, respectively. Conclusions: Biologically relevant GAs can be detected in pts with mRCC using blood and urine. Our methodology showed high concordance between both platforms. Consistent with prior reports, ctDNA assessment of pts with pretreated mRCC shows a higher frequency of TOR pathway alterations and a lower frequency of VHL mutation.

Identification of Somatic Mutations in Thirty-year-old Serum Cell-free DNA From Patients With Breast Cancer: A Feasibility Study

IntroductionThe aim of this study was to assess the feasibility of cell-free DNA (cfDNA) extraction and circulating tumor DNA sequencing in 30-year-old serum samples. Materials and MethodsWe evaluated serum samples from 52 patients with breast cancer, which were collected between 1983 and 1991, with correlating clinicopathologic data. cfDNA was extracted by using the QIAamp Circulating Nucleic Acid Extraction Kit (Qiagen). Of these 52 cfDNA samples, 10 were randomly selected and sequenced with the Oncomine Breast cfDNA Assay (A31183). In a second step, high-depth targeted sequencing of 15 additional cfDNA samples was performed using a custom Ampliseq Ion Torrent panel targeting breast cancer-related genes. ResultscfDNA extraction was successful in 52 (100%) of 52 patients with a total concentration of 0.2 to 54 ng/uL. A total of 24 cancer-specific mutations were found in 22 (88%) of the 25 samples undergoing sequencing. Of the 52 patients, 32 (62%) had died from breast cancer after a median follow-up of 7.9 years (interquartile range, 3.7-15.5 years). ConclusionThe present study shows that current next generation sequencing technology is sufficiently robust and specific to analyze 30-year-old serum. Therefore, longitudinal studies can be designed with storage of serum samples over many years, thereby obviating the need for timely and continuous cfDNA extraction and sequencing. The samples can be pooled and processed at once with the most modern technology available at the end of the study, when accumulation of events allows correlation of clinical outcomes with adequate power.

Neoantigen Tracking: Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing during Checkpoint Blockade Immunotherapy in Non‐Small Cell Lung Cancer (Adv. Sci. 9/2020)

Neoantigen Tracking: Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing during Checkpoint Blockade Immunotherapy in Non‐Small Cell Lung Cancer (Adv. Sci. 9/2020)

Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing during Checkpoint Blockade Immunotherapy in Non-Small Cell Lung Cancer.

The evolutionary dynamics of tumor‐associated neoantigens carry information about drug sensitivity and resistance to the immune checkpoint blockade (ICB). However, the spectrum of somatic mutations is highly heterogeneous among patients, making it difficult to track neoantigens by circulating tumor DNA (ctDNA) sequencing using “one size fits all” commercial gene panels. Thus, individually customized panels (ICPs) are needed to track neoantigen evolution comprehensively during ICB treatment. Dominant neoantigens are predicted from whole exome sequencing data for treatment‐naïve tumor tissues. Panels targeting predicted neoantigens are used for personalized ctDNA sequencing. Analyzing ten patients with non‐small cell lung cancer, ICPs are effective for tracking most predicted dominant neoantigens (80–100%) in serial peripheral blood samples, and to detect substantially more genes (18–30) than the capacity of current commercial gene panels. A more than 50% decrease in ctDNA concentration after eight weeks of ICB administration is associated with favorable progression‐free survival. Furthermore, at the individual level, the magnitude of the early ctDNA response is correlated with the subsequent change in tumor burden. The application of ICP‐based ctDNA sequencing is expected to improve the understanding of ICB‐driven tumor evolution and to provide personalized management strategies that optimize the clinical benefits of immunotherapies.

Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer

Little is known about mechanisms of resistance to poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy in patients with metastatic breast cancer and BRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure. We obtained tumor biopsies from metastatic breast cancer patients with BRCA1/2 deficiency before and after acquired resistance to PARPi or platinum chemotherapy. Whole exome sequencing was carried outon each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was carried out for functional assessment of intact homologous recombination (HR). Pre- and post-resistance tumor samples were sequenced from eight patients (four with BRCA1 and four with BRCA2 mutation; four treated with PARPi and four with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germline BRCA1 deficiency acquired genomic alterations anticipated to restore HR through increased DNA end resection: loss of TP53BP1 in one patient and amplification of MRE11A in another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of HR. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy. Genomic reversion in BRCA1/2 was the most commonly observed mechanism of resistance, occurring in four of eight patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

Studies Show Clinical Utility of Precision Medicine

As precision medicine has become increasingly important in cancer therapy, researchers have grappled with how to implement it in the clinic. Three studies demonstrate how different approaches to precision medicine-molecular profiling using individually customized combination therapy, RNA transcriptomics, and circulating tumor DNA sequencing-can be used to match patients to targeted treatments and trials.

Abstract P4-01-06: Comparison of tumor genotyping and cell-free circulating tumor DNA sequencing in metastatic breast cancer patients and their utility in the selection of matched therapy

Abstract Background: Oncogenic mutations are potential targets for therapeutic intervention in metastatic breast cancer (MBC). While tumor genotyping (TG) has been viewed as the gold standard for identifying oncogenic mutations, cell-free circulating tumor DNA (cfDNA) is emerging as an alternate technique. We previously reported the selection of matched therapy targeted to an actionable mutation based on either TG or cfDNA testing (Vidula N, ASCO, 2018). Therefore, we are now comparing TG and cfDNA results in MBC patients undergoing both tests to examine their relative utility in the selection of matched therapy. Methods: Patients with MBC at an academic institution who underwent both TG (Next Generation Sequencing/NGS, institutional platform, 104 gene assay) and cfDNA testing (NGS/Guardant360, 73 gene assay) between 1/2016-10/2017 were identified. A chart review was conducted to identify tumor subtype, demographics, treatment, TG and cfDNA results, and clinical outcomes. The relative utility of these tests in the selection of matched therapy was determined, and linked with clinical outcomes (progression-free survival and overall survival). Results: Thirty patients who underwent both TG and cfDNA testing were identified. The median age was 60 years, the majority (97%) had hormone receptor (HR) positive/HER2 negative disease, and most patients had recurrent disease (83.3%) at MBC diagnosis. The median number of therapies prior to obtaining either test was 1 (cfDNA range 0-9, TG range 0-8). The majority had simultaneous cfDNA and tumor genotyping testing (83.3%) versus sequential testing (16.7%). Twenty-four (80%) patients had actionable mutations detected by cfDNA compared to 19 (63.3%) patients with actionable mutations detected by TG. The median number of actionable mutations detected by cfDNA was 2 (range 0-11) compared with a median of 1 (range 0-4) detected by TG. Failure of TG occurred in 2 of 30 patients (6.7%) but no test failures were seen with cfDNA. Eleven of 30 patients (36.7%) had ≥ 1 concordant mutation via cfDNA and TG. Altogether, 12 out of 30 (40%) patients received matched therapy, 5 of which were based on cfDNA actionable mutations alone (ESR1, ERBB2, CCND1, and PIK3CA), and 7 based on cfDNA and TG results (ESR1, PIK3CA, STK11, and BRCA). Twelve of 24 (50%) patients with actionable cfDNA mutations went on to receive matched therapy compared with 7 of 19 (36.8%) patients with actionable TG results. Matched therapies included SERDs, inhibitors of CDK 4/6, PI3K, mTOR, HER2 directed therapy, and DNA damaging chemotherapy. The impact of matched therapy on survival outcomes will be presented at the meeting. Conclusions: In patients undergoing both TG and cfDNA testing, both tests identify a significant cohort of HR+ MBC patients with actionable mutations, with greater detection of actionable mutations by cfDNA. Greater application of matched therapy occurred via cfDNA, which independently informed the selection of matched therapies. Further research is needed to prospectively evaluate the clinical utility of blood based genotyping assays versus TG for patients with MBC. Citation Format: Vidula N, Juric D, Niemierko A, Spring L, Moy B, Malvarosa G, Yuen M, Habin K, Shin J, Peppercorn J, Isakoff S, Ellisen L, Iafrate AJ, Bardia A. Comparison of tumor genotyping and cell-free circulating tumor DNA sequencing in metastatic breast cancer patients and their utility in the selection of matched therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-06.

Liquid biopsy: a powerful tool to monitor trastuzumab resistance in HER2-positive metastatic gastric cancer

Human epidermal growth factor receptor 2 (HER2) overexpression/amplification affects about 6.1%–23.0% of gastric cancer patients [1, 2]. All major guidelines recommend HER2 testing to guide the selection for trastuzumab treatment in metastatic gastric cancer (mGC) [3], because doublet chemotherapy with trastuzumab significantly improved overall survival (OS) in the crucial trastuzumab for gastric cancer trial (the ToGA trial) [4]. However, the unsatisfactory gain of median progression-free survival (PFS) stresses important issues of intrinsic and acquired resistance to HER2-targeted therapies [4]. Currently, the underlying molecular mechanism of trastuzumab resistance in mGC is still unclear, and the strategies to real time monitor resistance-specific aberrations are urgently needed. As circulating tumor DNA (ctDNA) carries the same genomic alterations that are present in the primary tumor [5, 6], liquid biopsy-based ctDNA profiling offers a unique opportunity for serially monitoring treatment response in a non-invasive manner, and has opened the door for identification the mechanisms of drug resistance. In a study recently published in Gut, titled “Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer”, Wang et al. [7] demonstrated for the first time that longitudinal ctDNA sequencing could promisingly predict tumor shrinkage and progression in HER2 + mGC patients under trastuzumab treatment. Based on the detected molecular alterations from the ctDNA profiling, they further dissected various mechanisms of trastuzumab resistance (Fig. 1). Longitudinal circulating tumor DNA sequencing provides novel insights into gene alterations underlying trastuzumab resistance in HER2-positive metastatic gastric cancer. HER2+: HER2-positive; mGC: metastatic gastric cancer; ctDNA: circulating tumor DNA: Copy No.: copy number In this study, the authors sequenced 416 clinically relevant genes to determine the genomic profiles of GC patients. The molecular alterations, including copy number alterations, mutations and structural variations, detected in 78 paired plasma and tissue samples (46 from HER2+ patients and 32 from HER2− patients) were used to evaluate the consistency of detection between the “liquid” and “solid” biopsies. As expected, the alterations detected in plasma provided a good representation of the status in the tumor tissues. In particular, HER2 amplification status, the major marker of HER2 + GC, detected by plasma-based ctDNA sequencing was highly consistent with that by tissue-based sequencing and fluorescence in situ hybridisation. These findings indicated that plasma-based ctDNA profiling might provide insights into HER2 amplification in HER2 + GC. Further longitudinal analysis were perform in 97 time-course plasma samples collected from 24 HER2 + mGC patients to track the resistance during trastuzumab treatment. As compared to plasma carcinoembryonic antigen, the copy number variations (CNVs) of HER2 in ctDNA were better at tracking trastuzumab treatment response. Generally, the HER2 copy number decreased during treatment compared with baseline and levels of progressive disease (PD), indicating tumor shrinkage. Additionally, most patients with intrinsic resistance-related progression presented higher HER2 copy numbers at PD than baseline, while the patients with acquired resistance often had lower HER2 copy numbers at PD. From the same ctDNA profiles, the authors found evidence for reported mechanisms of trastuzumab resistance in 76.5% patients. They found that the patients with multiple resistance mechanisms (PIK3CA/R1/C3 and ERBB2/4 mutations) displayed much shorter PFS during trastuzumab treatment. These findings suggested that liquid biopsy-based ctDNA profiling can effectively capture the heterogeneity information about acquired resistance. Furthermore, they also observe several novel resistance mechanisms. One of them is the ERBB4 S774G mutation. This novel mutation can increase the sensitivity to trastuzumab treatment, suggesting for the first time that the mutation of ERBB4 play a critical role in mediating resistance to ERBB2 inhibitors in HER2 + GC. Another novel resistance mechanism is the emergence of NF1 mutations. Using in vitro and in vivo experiments, the authors demonstrated that NF1 was a resistance-related gene and could be used as a potential resistance marker of trastuzumab. NF1 lost (mutation or deletion) contributes to driving resistance of HER2-targeted therapy via elevating pMEK/ERK activation, whereas the combination of HER2 and MEK/ERK inhibitors might overcome trastuzumab resistance. This study has some obvious limitations. First, other rare genomic or even nongenomic mechanisms of trastuzumab resistance may have been missed by the 416-gene panel. Second, the relatively small number of patient enrolled does not allow drawing definitive conclusion on the predictive value of this liquid biopsy approach. Nevertheless, the study provides helpful information to monitor the treatment response of trastuzumab and develop therapeutic strategies for the HER2 + mGC patients with trastuzumab resistance. The author read and approved the final manuscript. Not applicable. The author declares no competing interests. Not applicable. Not applicable. Not applicable. Not applicable.