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BACKGROUND & AIMS: Patient-derived organoids (PDOs) are promising tumor avatars that could enable ex vivo drug tests to personalize patients' treatments in the frame of functional precision oncology. However, clinical evidence remains scarce. This study aims to evaluate whether PDOs can be implemented in clinical practice to benefit patients with advanced refractory pancreatic ductal adenocarcinoma (PDAC). METHODS: During 2021 to 2022, 87 patients were prospectively enrolled in an institutional review board-approved protocol. Inclusion criteria were histologically confirmed PDAC with the tumor site accessible. A panel of 25 approved antitumor therapies (chemogram) was tested and compared to patient responses to assess PDO predictive values and map the drug sensitivity landscape in PDAC. RESULTS: Fifty-four PDOs were generated from 87 pretreated patients (take-on rate, 62%). The main PDO mutations were KRAS (96%), TP53 (88%), and CDKN2A/B (22%), with a 91% concordance rate with their tumor of origin. The mean turnaround time to chemogram was 6.8 weeks. In 91% of cases, ≥1 hit was identified (gemcitabine (n = 20 of 54), docetaxel (n = 18 of 54), and vinorelbine (n = 17 of 54), with a median of 3 hits/patient (range, 0-12). Our cohort included 34 evaluable patients with full clinical follow-up. We report a chemogram sensitivity of 83.3% and specificity of 92.9%. The overall response rate and progression-free survival were higher when patients received a hit treatment as compared to patients who received a nonhit drug (as part of routine management). Finally, we leveraged our PDO collection as a platform for drug validation and combo identification. We tested anti-KRASG12D (MRTX1133), alone or combined, and identified a specific synergy with anti-EGFR therapies in KRASG12D variants. CONCLUSIONS: We report the largest prospective study aiming at implementing PDO-based functional precision oncology and identify very robust predictive values in this clinical setting. In a clinically relevant turnaround time, we identify putative hits for 91% of patients, providing unexpected potential survival benefits in this very aggressive indication. Although this remains to be confirmed in interventional precision oncology trials, PDO collection already provides powerful opportunities for drugs and combinatorial treatment development.
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BACKGROUND: Molecular characterization has significantly improved the management of advanced endometrial cancer (EC). It distinguishes four molecular subclasses associated with prognosis and personalized therapeutic strategies. This study assesses the clinical utility of cell-free DNA (cfDNA) profiling in EC to identify targetable alterations. METHODS: Women with metastatic or recurrent EC were prospectively recruited within the framework of the STING trial (NCT04932525), during which cfDNA was analyzed. Genomic alterations were identified with the FoundationOne CDx assay. Each molecular report underwent review by a molecular tumor board. Alterations were categorized via the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT). RESULTS: A total of 61 patients were enrolled. The median age was 66.9 years, with 43% presenting frontline metastatic disease. All histologic subgroups were represented. Notably, 89% of patients yielded informative cfDNA analysis. Six tumors were classified with deficient mismatch repair/microsatellite instability (11%) and 37 as TP53 gene mutant (67%), and 12 had nonspecific molecular profiles (22%). Molecular classification based on liquid biopsy showed 87.5% accuracy in correlating with tissue results. Moreover, 65% of cases exhibited ≥1 actionable alteration, including 25% ESCAT I alterations and 13% ESCAT II alterations. Consequently, 16% of patients received a molecularly matched therapy, and presented with a 56% response rate and median progression-free survival of 7.7 months. CONCLUSIONS: cfDNA sequencing in EC is a feasible approach that produces informative results in 89% of cases and accurately categorizes patients into the main molecular subclasses. It also reveals multiple actionable alterations, which offers the potential for personalized therapeutic strategies.
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BACKGROUND: Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. METHODS: Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. RESULTS: Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45(-) cells, cytomorphological staining, then scanning and analysis of CD45(-) cell phenotypical and cytomorphological characteristics. CD45(-) cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 µm(2). The second assay sequentially combined fluorescent staining, automated selection of CD45(-) cells, FISH scanning on CD45(-) cells, then analysis of CD45(-) cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 µm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. CONCLUSIONS: The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic.
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Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/metabolismo , Quinase do Linfoma Anaplásico , Automação Laboratorial , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Separação Celular , Forma Celular , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Microscopia de Fluorescência , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Biliary tract cancers (BTCs) are heterogeneous malignancies with dismal prognosis due to tumor aggressiveness and poor response to limited current therapeutic options. Tumor exome profiling has allowed to successfully establish targeted therapeutic strategies in the clinical management of cholangiocarcinoma (CCA). Still, whether liquid biopsy profiling could inform on BTC biology and patient management is unknown. In order to test this and generate novel insight into BTC biology, we analyzed the molecular landscape of 128 CCA patients, using a 394-gene NGS panel (Foundation Medicine). Among them, 32 patients had matched circulating tumor (ct) DNA and tumor DNA samples, where both samples were profiled. In both tumor and liquid biopsies, we identified an increased frequency of alterations in genes involved in genome integrity or chromatin remodeling, including ARID1A (15%), PBRM1 (9%), and BAP1 (14%), which were validated using an in-house-developed immunohistochemistry panel. ctDNA and tumor DNA showed variable concordance, with a significant correlation in the total number of detected variants, but some heterogeneity in the detection of actionable mutations. FGFR2 mutations were more frequently identified in liquid biopsies, whereas KRAS alterations were mostly found in tumors. All IDH1 mutations detected in tumor DNA were also identified in liquid biopsies. These findings provide novel insights in the concordance between the tumor and liquid biopsies genomic landscape in a large cohort of patients with BTC and highlight the complementarity of both analyses when guiding therapeutic prescription.
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BACKGROUND: Genomic studies have identified new subsets of aggressive prostate cancer (PCa) with poor prognosis (eg, neuroendocrine prostate cancer [NEPC], PCa with DNA damage response [DDR] alterations, or PCa resistant to androgen receptor pathway inhibitors [ARPIs]). Development of novel therapies relies on the availability of relevant preclinical models. OBJECTIVE: To develop new preclinical models (patient-derived xenograft [PDX], PDX-derived organoid [PDXO], and patient-derived organoid [PDO]) representative of the most aggressive variants of PCa and to develop a new drug evaluation strategy. DESIGN, SETTING, AND PARTICIPANTS: NEPC (n = 5), DDR (n = 7), and microsatellite instability (MSI)-high (n = 1) PDXs were established from 51 patients with metastatic PCa; PDXOs (n = 16) and PDOs (n = 6) were developed to perform drug screening. Histopathology and treatment response were characterized. Molecular profiling was performed by whole-exome sequencing (WES; n = 13), RNA sequencing (RNA-seq; n = 13), and single-cell RNA-seq (n = 14). WES and RNA-seq data from patient tumors were compared with the models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Relationships with outcome were analyzed using the multivariable chi-square test and the tumor growth inhibition test. RESULTS AND LIMITATIONS: Our PDXs captured both common and rare molecular phenotypes and their molecular drivers, including alterations of BRCA2, CDK12, MSI-high status, and NEPC. RNA-seq profiling demonstrated broad representation of PCa subtypes. Single-cell RNA-seq indicates that PDXs reproduce cellular and molecular intratumor heterogeneity. WES of matched patient tumors showed preservation of most genetic driver alterations. PDXOs and PDOs preserve drug sensitivity of the matched tissue and can be used to determine drug sensitivity. CONCLUSIONS: Our models reproduce the phenotypic and genomic features of both common and aggressive PCa variants and capture their molecular heterogeneity. Successfully developed aggressive-variant PCa preclinical models provide an important tool for predicting tumor response to anticancer therapy and studying resistance mechanisms. PATIENT SUMMARY: In this report, we looked at the outcomes of preclinical models from patients with metastatic prostate cancer enrolled in the MATCH-R trial (NCT02517892).
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Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Animais , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Modelos Animais de DoençasRESUMO
PURPOSE: The androgen receptor axis inhibitors (ARPI; e.g., enzalutamide, abiraterone acetate) are administered in daily practice for men with metastatic castration-resistant prostate cancer (mCRPC). However, not all patients respond, and mechanisms of both primary and acquired resistance remain largely unknown. EXPERIMENTAL DESIGN: In the prospective trial MATCH-R (NCT02517892), 59 patients with mCRPC underwent whole-exome sequencing (WES) and/or RNA sequencing (RNA-seq) of samples collected before starting ARPI. Also, 18 patients with mCRPC underwent biopsy at time of resistance. The objectives were to identify genomic alterations associated with resistance to ARPIs as well as to describe clonal evolution. Associations of genomic and transcriptomic alterations with primary resistance were determined using Wilcoxon and Fisher exact tests. RESULTS: WES analysis indicated that no single-gene genomic alterations were strongly associated with primary resistance. RNA-seq analysis showed that androgen receptor (AR) gene alterations and expression levels were similar between responders and nonresponders. RNA-based pathway analysis found that patients with primary resistance had a higher Hedgehog pathway score, a lower AR pathway score and a lower NOTCH pathway score than patients with a response. Subclonal evolution and acquisition of new alterations in AR-related genes or neuroendocrine differentiation are associated with acquired resistance. ARPIs do not induce significant changes in the tumor transcriptome of most patients; however, programs associated with cell proliferation are enriched in resistant samples. CONCLUSIONS: Low AR activity, activation of stemness programs, and Hedgehog pathway were associated with primary ARPIs' resistance, whereas most acquired resistance was associated with subclonal evolution, AR-related events, and neuroendocrine differentiation. See related commentary by Slovin, p. 4323.
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Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Proteínas Hedgehog , Estudos Prospectivos , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/genética , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Genômica , NitrilasRESUMO
BACKGROUND: Genomic stratification may help improve the management of patients with metastatic urothelial cancer (mUC), given the recent identification of targetable alterations. However, the collection of tissue samples remains challenging. Here, we assessed the clinical utility of plasma circulating tumour DNA (ctDNA) sequencing in these patients. METHODS: Patients with mUC were prospectively enroled in the STING trial (NCT04932525), in which ctDNA was profiled using the Foundation One Liquid CDx Assay (324 genes, blood tumour mutational burden [bTMB], microsatellite instability status). Each genomic report was reviewed by a multidisciplinary tumor board (MTB). RESULTS: Between January 2021 and June 2022, 140 mUC patients underwent molecular profiling. The median time to obtain the assay results was 20 days ((confidence interval) CI95%: [20,21]). The ctDNA analysis reproduced the somatic genomic landscape of previous tissue-based cohorts. Concordance for serial ctDNA samples was strong (r = 0.843 CI95%: [0.631-0.938], p < 0.001). At least one actionable target was detected in 63 patients (45%) with a total of 35 actionable alterations, including bTMB high (≥10 mutations/Mb) (N = 39, 21.1%), FGFR3 (N = 20, 10.8%), and Homologous recombination deï¬ciency (HRD) alterations (N = 14, 7.6%). MTB recommended matched therapy in 63 patients (45.0%). Eight patients (5.7%) were treated, with an overall response rate of 50% (CI95%: 15.70-84.30) and a median progression-free survival (PFS) of 5.2 months (CI95%: 4.1 - NR). FGFR3 alterations were associated with a shorter PFS in patients treated with immunotherapy. CONCLUSION: Overall, we demonstrated that genomic profiling with ctDNAs in mUC is a reliable and feasible approach for the timely initiation of genotype-matched therapies.
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DNA Tumoral Circulante , Neoplasias , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Tumoral Circulante/genética , Biomarcadores Tumorais/genética , Genômica/métodos , MutaçãoRESUMO
Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.
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Apoptose , Bacillus cereus/patogenicidade , Proteínas de Bactérias/toxicidade , Proteínas Hemolisinas/toxicidade , Macrófagos/microbiologia , Fatores de Virulência/toxicidade , Animais , Bacillus cereus/genética , Proteínas de Bactérias/genética , Bombyx , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Deleção de Genes , Proteínas Hemolisinas/genética , Humanos , Larva/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Virulência , Fatores de Virulência/genéticaRESUMO
INTRODUCTION: Although the role of epigenetic alterations in oncogenesis has been well studied, their prevalence in metastatic solid tumours is still poorly described. We therefore aimed at: (i) describing the presence of epigenetic gene alterations (EGA) - defined by an alteration in a gene encoding an epigenetic regulator; and (ii) evaluating their relationship with clinical characteristics and outcome in patients (pts) included in prospective molecular profiling trials. MATERIALS AND METHODS: On-purpose tumour biopsies from pts with metastatic solid tumours enrolled in the Gustave Roussy-sponsored MOSCATO (NCT01566019) and MATCHR (NCT02517892) trials were molecularly profiled using whole exome sequencing (WES). Alterations in 176 epigenetic genes were assessed and classified as pathogenic variants (PV) or non-pathogenic variants by a molecular tumour board. Clinical characteristics and outcome were collected. RESULTS: Between Dec 2011 and Oct 2016, WES was successfully performed in 292 pts presenting various solid tumours. We found 496 epigenetic gene alterations in 134 patients (49%), including 237 pathogenic variants in 86 patients; 63 tumour samples (47%) presented ≥3 EGAs. The median number of previous treatment lines was 3 (1-10). The most frequently altered genes were KMT2D and KMT2C (16% each), ARID1A and SETD2 (10% each) and KMT2A (8%).; 31% of EGA co-occurred with a driver gene alteration (p < 0.001). Outcome was not correlated with the presence of EGA. CONCLUSIONS: Epigenetic alterations occur frequently in metastatic solid tumours. With the current development of epigenetic modifiers, they increasingly represent actionable targets. Such genes should now be systematically analysed in molecular profiling studies.
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Neoplasias , Medicina de Precisão , Biomarcadores Tumorais/genética , Epigênese Genética , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão/métodos , Sequenciamento do ExomaRESUMO
FDA-approved next-generation sequencing assays based on cell-free DNA offers new opportunities in a molecular-tumor-board context thanks to the noninvasiveness of liquid biopsy, the diversity of analyzed parameters and the short turnaround time. It gives the opportunity to study the heterogeneity of the tumor, to elucidate complex resistance mechanisms and to adapt treatment strategies. However, lowering the limit of detection and increasing the panels' size raise new questions in terms of detection of incidental germline alterations, occult malignancies and clonal hematopoiesis of indeterminate potential mutations. In this review, after a technological discussion and description of the common problematics encountered, we establish recommendations in properly using these FDA-approved tests in a molecular-tumor-board context.
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Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias , DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Neoplasias/genética , Estados Unidos , United States Food and Drug AdministrationRESUMO
INTRODUCTION: Despite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression. METHODS: Patients with EGFR-mutated metastatic NSCLC (N = 62) with tissue and plasma biopsies at EGFR TKI progression between January 2015 and June 2019, at a French hospital and optionally before progression, were identified from the prospective MATCH-R study (NCT02517892). Postprogression biopsy samples were analyzed for gene fusions using targeted gene panel sequencing, whole-exome sequencing, RNA sequencing, and comparative genomic hybridization array. RESULTS: Six gene fusions were detected in tumor progression biopsies under an EGFR TKI from 62 consecutive patients (9.7%) with EGFR-mutated advanced NSCLC. Among 31 patients progressing to first- or second-generation EGFR TKIs, one (3%) had an Eukaryotic translation initiation factor 4 gamma 2-GRB2 associated binding protein 1 (EIF4G2-GAB1) fusion. Among 31 patients progressing to the third-generation osimertinib, five (16%) presented oncogene fusions of fibroblast growth factor receptor 3-transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3) (n = 2), kinesin family member 5B-Ret proto-oncogene (KIF5B-RET) (n = 1), striatin-anaplastic lymphoma kinase (STRN-ALK) (n = 1), and zinc finger DHHC-Type palmitoyltransferase 20-Thr790Met (ZDHHC20-BRAF) (n = 1) transcripts. Out of two patients that received osimertinib at first-line, one acquired an FGFR3-TACC3 fusion at progression. In all patients, fusions co-occurred with the original activating EGFR mutation; however, among four patients with an acquired T790M mutation, three (75%) lost the T790M mutation. CONCLUSIONS: Oncogenic fusions at the time of EGFR TKI resistance were identified at a relatively high frequency, mainly after the third-generation TKI osimertinib. Patients progressing to EGFR TKIs may have a new opportunity for targeted therapy when oncogenic fusions are identified.
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INTRODUCTION: In patients with oncogene-addicted NSCLC and isolated central nervous system progression (iCNS), tissue biopsy is challenging, and the clinical utility of plasma liquid biopsy (i.e., circulating tumor DNA [ctDNA]) is unknown. METHODS: Patients with advanced NSCLC with known baseline genomic alteration (GA) (EGFR, ALK, BRAF, KRAS, HER2, ROS1, MET, PIK3CA, STK11, TP53) on tissue were divided into three groups on the basis of their disease progression pattern: iCNS, extra-CNS only (noCNS), or both (cCNS). All patients with available plasma ctDNA were included and were analyzed by next-generation sequencing InVisionFirst-Lung. ctDNA was considered positive if at least one GA was detected. Cell-free tumor DNA was analyzed in cerebrospinal fluid when available. RESULTS: Out of 517 patients screened, 247 were included: 54 had iCNS, 99 had noCNS, and 94 had cCNS progressive disease (64, 128, and 110 ctDNA samples, respectively). CtDNA was positive in 52% iCNS versus 84% in noCNS and 92% in cCNS (p < 0.00001), with lower detection of driver (37% versus 77% and 73%, respectively) and resistance alterations (6% versus 45% and 44%). Patients with iCNS and positive ctDNA were more at risk of extra-CNS progression (32% versus 7%, p = 0.026). In 12 patients with iCNS, ctDNA was positive in six (50%) plasma and in 10 (83%) paired cerebrospinal fluid (p = 0.193). CONCLUSIONS: Although tagged amplicon-based next-generation sequencing has high detection rates of GA in plasma ctDNA in patients with NSCLC with extra-CNS disease, detection rate of GAs (52%) is lower in the subset of patients with iCNS disease. Complementary tests such as cerebrospinal fluid cell-free DNA may be useful. Further evidence would be beneficial to understand the genomic landscape in patients with NSCLC and iCNS.
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DNA Tumoral Circulante , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Sistema Nervoso Central , DNA Tumoral Circulante/genética , Progressão da Doença , Humanos , Neoplasias Pulmonares/genética , Mutação , Oncogenes , Proteínas Tirosina Quinases , Proteínas Proto-OncogênicasRESUMO
Unravelling the biological processes driving tumour resistance is necessary to support the development of innovative treatment strategies. We report the design and feasibility of the MATCH-R prospective trial led by Gustave Roussy with the primary objective of characterizing the molecular mechanisms of resistance to cancer treatments. The primary clinical endpoints consist of analyzing the type and frequency of molecular alterations in resistant tumours and compare these to samples prior to treatment. Patients experiencing disease progression after an initial partial response or stable disease for at least 24 weeks underwent a tumour biopsy guided by CT or ultrasound. Molecular profiling of tumours was performed using whole exome sequencing, RNA sequencing and panel sequencing. At data cut-off for feasibility analysis, out of 333 inclusions, tumour biopsies were obtained in 303 cases (91%). From these biopsies, 278 (83%) had sufficient quality for analysis by high-throughput next generation sequencing (NGS). All 278 samples underwent targeted NGS, 215 (70.9%) RNA sequencing and 222 (73.2%) whole exome sequencing. In total, 163 tumours were implanted in NOD scid gamma (NSG) or nude mice and 54 patient-derived xenograft (PDX) models were established, with a success rate of 33%. Adverse events secondary to invasive tumour sampling occurred in 24 patients (7.6%). Study recruitment is still ongoing. Systematic molecular profiling of tumours and the development of patient-derived models of acquired resistance to targeted agents and immunotherapy is feasible and can drive the selection of the next therapeutic strategy.
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PURPOSE: The limited knowledge on the molecular profile of patients with BRAF-mutant non-small cell lung cancer (NSCLC) who progress under BRAF-targeted therapies (BRAF-TT) has hampered the development of subsequent therapeutic strategies for these patients. Here, we evaluated the clinical utility of circulating tumor DNA (ctDNA)-targeted sequencing to identify canonical BRAF mutations and genomic alterations potentially related to resistance to BRAF-TT, in a large cohort of patients with BRAF-mutant NSCLC. EXPERIMENTAL DESIGN: This was a prospective study of 78 patients with advanced BRAF-mutant NSCLC, enrolled in 27 centers across France. Blood samples (n = 208) were collected from BRAF-TT-naïve patients (n = 47), patients nonprogressive under treatment (n = 115), or patients at disease progression (PD) to BRAF-TT (24/46 on BRAF monotherapy and 22/46 on BRAF/MEK combination therapy). ctDNA sequencing was performed using InVisionFirst-Lung. In silico structural modeling was used to predict the potential functional effect of the alterations found in ctDNA. RESULTS: BRAFV600E ctDNA was detected in 74% of BRAF-TT-naïve patients, where alterations in genes related with the MAPK and PI3K pathways, signal transducers, and protein kinases were identified in 29% of the samples. ctDNA positivity at the first radiographic evaluation under treatment, as well as BRAF-mutant ctDNA positivity at PD were associated with poor survival. Potential drivers of resistance to either BRAF-TT monotherapy or BRAF/MEK combination were identified in 46% of patients and these included activating mutations in effectors of the MAPK and PI3K pathways, as well as alterations in U2AF1, IDH1, and CTNNB1. CONCLUSIONS: ctDNA sequencing is clinically relevant for the detection of BRAF-activating mutations and the identification of alterations potentially related to resistance to BRAF-TT in BRAF-mutant NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , DNA Tumoral Circulante/genética , Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular/métodos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , DNA Tumoral Circulante/análise , Seguimentos , Genômica/métodos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Prognóstico , Estudos Prospectivos , Taxa de SobrevidaRESUMO
PURPOSE: Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting. EXPERIMENTAL DESIGN: Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance. RESULTS: Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors. CONCLUSIONS: This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.
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Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Aminopiridinas , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Feminino , Rearranjo Gênico , Humanos , Lactamas , Lactamas Macrocíclicas/uso terapêutico , Estudos Longitudinais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Neurofibromina 2/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Modern oncology requires precise tumor assessment to drive effective therapies. Image-guided biopsies are the current standard of care to characterize molecular alterations safely and effectively, but have inherent limitations due to tumor heterogeneity and accessibility, as well as from procedure related risks. Advancements in radiomics analysis provide the potential to retrieve useful incremental information to characterize molecular alterations from standard imaging data in a cost-effective and non-invasive manner, but currently suffers from lack of validation and standardization. The combination of techniques may provide the optimal solution for patient-tailored care, ultimately through the development of accurate and reliable virtual biopsy. In the advancement towards that goal, image-guided biopsy can prove radiomic suspicions and conversely radiomics can guide image-guided biopsy to improve tissue yield. Joint development of these two methods will improve cancer understanding and patient outcomes.
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Biópsia Guiada por Imagem/métodos , Medicina de Precisão/métodos , Radiometria/métodos , HumanosRESUMO
PURPOSE: To evaluate efficacy, complications and preprocedural risk factors for percutaneous image-guided core needle biopsy of malignant tumours for genomic tumour analysis. MATERIALS AND METHODS: Procedural data for core biopsies performed at a single centre for the MOSCATO-01 clinical trial were prospectively recorded between December 2011 and March 2016. Data assessed included patient demographics, tumour characteristics, procedural outcomes and complications. RESULTS: A total of 877 biopsies were performed under computed tomography (38.4%) or ultrasound guidance (61.6%) for tumours in the liver (n = 363), lungs (n = 229), lymph nodes (n = 138), bones (n = 15) and other miscellaneous sites (n = 124). Each biopsy harvested a mean 4.4 samples [1-15], with adequate tumour yield for genomic analysis in 95.3% of cases. Procedural complications occurred in 89 cases (10.1%), with minor grade I complications in 59 (66.3%); grade II in 16 (18%) and grade III in 14 (15.7%). No grade IV complications and no procedure-related death occurred. The most common complications were pneumothorax (51/89, 57.3%), haemorrhage (24/89, 27%) and pain (8/89, 8.9%). Predictive factors for complications by univariate analysis included biopsied organ (lung vs other), sample number, prone position, lesion size, lesion depth and biopsy approach. By multivariate analysis, only pulmonary biopsy was a significant risk factor (odds ratio = 27.23 [4.93-242.76], p < 0.01). CONCLUSION: Percutaneous image-guided core needle biopsy in cancer patients provides an effective method to obtain molecular screening samples, with an overall low complication rate. Lung mass biopsies present a higher risk of complication, although complications are manageable by minimally invasive techniques without prolonged sequelae.
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Biópsia Guiada por Imagem/métodos , Neoplasias/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
High-throughput genomic analyses may improve outcomes in patients with advanced cancers. MOSCATO 01 is a prospective clinical trial evaluating the clinical benefit of this approach. Nucleic acids were extracted from fresh-frozen tumor biopsies and analyzed by array comparative genomic hybridization, next-generation sequencing, and RNA sequencing. The primary objective was to evaluate clinical benefit as measured by the percentage of patients presenting progression-free survival (PFS) on matched therapy (PFS2) 1.3-fold longer than the PFS on prior therapy (PFS1). A total of 1,035 adult patients were included, and a biopsy was performed in 948. An actionable molecular alteration was identified in 411 of 843 patients with a molecular portrait. A total of 199 patients were treated with a targeted therapy matched to a genomic alteration. The PFS2/PFS1 ratio was >1.3 in 33% of the patients (63/193). Objective responses were observed in 22 of 194 patients (11%; 95% CI, 7%-17%), and median overall survival was 11.9 months (95% CI, 9.5-14.3 months).Significance: This study suggests that high-throughput genomics could improve outcomes in a subset of patients with hard-to-treat cancers. Although these results are encouraging, only 7% of the successfully screened patients benefited from this approach. Randomized trials are needed to validate this hypothesis and to quantify the magnitude of benefit. Expanding drug access could increase the percentage of patients who benefit. Cancer Discov; 7(6); 586-95. ©2017 AACR.See related commentary by Schram and Hyman, p. 552This article is highlighted in the In This Issue feature, p. 539.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Resultado do Tratamento , Adulto JovemRESUMO
The duration and magnitude of clinical response are unpredictable in ALK-rearranged non-small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543-13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222-30. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Receptores Proteína Tirosina Quinases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Crizotinibe , Variações do Número de Cópias de DNA/genética , Intervalo Livre de Doença , Feminino , Rearranjo Gênico/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/patologia , Prognóstico , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Receptores Proteína Tirosina Quinases/genéticaRESUMO
INTRODUCTION: MOSCATO-01 is a molecular triage trial based on on-purpose tumour biopsies to perform molecular portraits. We aimed at identifying factors associated with high tumour cellularity. MATERIAL AND METHODS: Tumour cellularity (percentage of tumour cells in samples defined at pathology) was evaluated according to patient characteristics, target lesion characteristics, operators' experience and biopsy approach. RESULTS: Among 460 patients enrolled between November, 2011 and March, 2014, 334 patients (73%) had an image-guided needle biopsy of the primary tumour (N = 38) or a metastatic lesion (N = 296). Biopsies were performed on liver (N = 127), lung (N = 72), lymph nodes (N = 71), bone (N = 11), or another tumour site (N = 53). Eighteen patients (5%) experienced a complication: pneumothorax in 10 patients treated medically, and haemorrhage in 8, requiring embolisation in 3 cases. Median tumour cellularity was 50% (interquartile range, 30-70%). The molecular analysis was successful in 291/334 cases (87%). On-going chemotherapy, tumour origin (primary versus metastatic), lesion size, tumour growth rate, presence of necrosis on imaging, standardised uptake value, and needle size were not statistically associated with cellularity. Compared to liver or lung biopsies, cellularity was significantly lower in bone and higher in other sites (P < 0.0001). Cellularity significantly increased with the number of collected samples (P < 0.0001) and was higher in contrast-enhanced ultrasound-guided biopsies (P < 0.02). In paired samples, cellularity in central samples was lower than in peripheral samples in 85, equal in 68 and higher in 89 of the cases. CONCLUSION: Image-guided biopsy is feasible and safe in cancer patients for molecular screening. Imaging modality, multiple sampling of the lesion, and the organ chosen for biopsy were associated with higher tumour cellularity.