Liquid biopsies to optimize therapeutic efficacy in unresponsive lung cancer patients

KEYWORDS:

• Lung cancer
• Pharmacogenomics
• Liquid Biopsy
• Intratumor Heterogeneity

Lung cancer represents the leading cause of cancer mortality worldwide [1]. Non small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung neoplasms and comprises different histotypes, including adenocarcinoma, squamous cell carcinoma, and large-cell lung cancer. Molecularly targeted therapies have radically changed treatment approach and prognosis of a subset of patients with oncogene-addicted tumors. However, long-term efficacy of these agents is limited by development of acquired resistance in almost all patients [2,3]. Recently, the use of immune checkpoint inhibitors has further expanded the therapeutic landscape for lung cancer, with efficacy observed in both squamous and non-squamous NSCLC patients [4]. For a large proportion of patients, however, cytotoxic chemotherapy, which is considered a suboptimal therapeutic ‘one-size fits all’ approach, remains the standard of care. The opportunity to select patients most likely responsive based on the presence of tumor-specific predictive biomakers is the mainstay of targeted therapies, includingepidermal growth factor receptor (EGFR)- and anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs). Therefore, molecular profiling of tumors at baseline and at disease progression emerges as a crucial step in the treatment decision-making process. However, the main pitfall to these treatments relies on tumor genetic heterogeneity. The use of large-scale sequencing analyses has indeed recently allowed a deep characterization of the molecular portraits of several tumor types, revealing their complexity and an extensive heterogeneity between individual tumors [5]. Moreover, multiregion sequencing of one or more neoplastic lesions has revealed genetic intratumor heterogeneity, defined as the coexistence of different cell populations with distinct molecular features within the same tumor or different lesions, e.g. primary and metastatic sites, from the same patient. Information deriving from molecular profiling of multiple regions of tumors can be useful to track its dynamic evolution over time (so-called phylogenetic reconstruction) and distinguish between clonal alterations, representing earlier events present in all tumor cells, and heterogeneous alterations present instead in a minority of cellular subclones [5]. The heterogeneity can be explained as a result of both (epi)genetic variability and the selection of distinct cellular subpopulations under the pressure of external factors, including targeted therapies. Indeed, the genomics of a tumor may vary not only in space, but also over time (namely ‘temporal heterogeneity’), thus likely explaining the acquisition of resistance to treatments, with the best example being represented by the development of resistance to EGFR TKIs. The identification of a secondary, resistance mutation within the TK domain of EGFR, the T790M, is crucial since it can offer the possibility of treatment with mutant-specific TKIs, including osimertinib, which has further improved the prognosis of these molecular subgroups of patients [6].

Recognition of intratumor heterogeneity is crucial since it may impact on tumor biopsy strategy, characterization of actionable targets, optimal treatment strategies selection, and drug resistance. In a landmark study, whole-exome multiregion sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites showed a significant intratumor heterogeneity regarding mutations in key oncogenes, including mammalian target of rapamycin (mTOR), and tumor suppressor genes, including SETD2 and PTEN, gene expression signatures, allelic composition, and ploidy profiling [7]. These results suggest the potential of tumor heterogeneity in conditioning therapeutic failure. Recent reports have shown that lung cancers are characterized by substantial heterogeneity. Indeed, in a study performing whole-exome multiregion sequencing on 48 tumor regions from 11 localized, resected lung adenocarcinomas, 76% of all mutations and 20 of 21 known cancer gene mutations were identified in all regions of individual tumors, suggesting these represent early events in tumorigenesis [8]. All patients who had relapsed after surgery showed significantly larger fractions of subclonal mutations in their primary tumors than those who did not, suggesting these subclonal mutations may also be involved in cancer progression. In a similar study, whole-exome or whole-genome sequencing of 25 tumor regions deriving from distinct tumor samples from 7 patients with resected lung cancer, all patients had mutations in at least 1 but not all regions sequenced, highlighting a spatial heterogeneity. Interestingly, in one region there was the presence of different activated oncogenes, including potentially druggable genomic targets, thus likely explaining a potential different treatment sensitivity depending of the molecular features of these cellular subclones [9].

Due to intratumor heterogeneity, a single tumor biopsy may be inadequate to portray the whole genomics profile and miss potential targetable gene mutations because of selection bias. Moreover, it is rarely feasible to obtain serial biopsies from primary tumor and metastatic sites to track tumor dynamics over the course of treatment and monitor the emergence of resistant subclones. This is mainly because most diagnostic procedures are invasive, potentially risky, and not acceptable for the patients.

In recent years, liquid biopsy has emerged as be a viable surrogate for tumor tissue for noninvasive assessment of tumor genetic and epigenetic alterations. Liquid biopsies refer to different tumor-derived components that can be isolated by blood, or even other body fluids, including circulating cell-free tumor DNA, cell-free tumor RNA, exosomes, tumor-educated platelets (TEP), and circulating tumor cells (CTCs) [10]. Liquid biopsy can offer several advantages compared to tissue biopsy, including the possibility to capture tumor heterogeneity and provide a wider representation of the entire genomic landscape, including primary tumor and multiple metastatic sites. In addition, the feasibility to collect multiple specimens at various points before and during treatment can be exploited to monitor tumor genomic changes over time and the emergence of heteregenous mechanisms of resistance [11]. Liquid biopsy has already been integrated into clinical practice in lung cancer, where circulating cell-free tumor (ctDNA) genotyping is currently used to analyze predictive biomarkers, mainly EGFR sensitizing and T790M mutations, as surrogate of tumor tissue profiling, to select patients for EGFR TKI therapy [12]. The spread of studies on liquid biopsies has proceeded in parallel with development of newer sensitive blood-based assays to test ctDNA at very low concentrations for most genomic abnormalities. These include polymerase chain reaction (PCR)-based techniques, including digital PCR, droplet digital PCR, peptide nucleic acid clamp-based PCR assay (Taqman assay), beads, emulsions, amplification and magnetics (BEAMing) and next-generation sequencing technologies [13]. Longitudinal collection and analysis of ctDNA in EGFR-mutated lung cancer patients during treatment with EGFR TKIs has revealed the acquisition by cells of heterogeneous mechanisms of resistance. Indeed, continuous administration of third-generation EGFR TKIs, such as osimeritinib or rociletinib, induces selection of different molecularly distinct subclones characterized by secondary genomic alterations in EGFR or EGFR-independent molecular mechanisms. The best characterized mechanism of resistance to osimertinib is the acquired mutation in C797S, with its allelic context defining sensitivity to subsequent treatments [6,14–16]. So, therapeutic decisions should be oriented by underlying molecular features of resistant cells. As we have commented, other biological sources can be used as liquid biopsy. Indeed, a combined analysis of EGFR genotyping in matched blood and urine by a highly sensitive and quantitative assay using next-generation sequencing was demonstrated to improve the chance of T790M mutation detection in the TIGER-X study of rociletinib [17], which is no longer in clinical development. Beyond ctDNA, CTCs and other tumor-derived materials, including exosomes and TEPs [18,19], have been shown to provide prognostic and diagnostic information and are currently extensively being investigated for several potential clinical applications. In this context, TEPs have demonstrated high specificity as nonnvasive source to assess EML4-ALK rearrangements detection and have proven usefulness in predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based exclusively on radiographic imaging alone [20].

The large interstudy variability of patient populations, sample size, and methodologies used to assess the different biomarkers through liquid biopsies underline the need for more robust data in order they could ultimately be integrated into clinical practice.

1. Expert opinion

Lung cancer treatment landscape has strikingly evolved over the last years mainly because of the introduction for clinical use of molecularly targeted agents and, more recently, of the immune checkpoint inhibitors targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) pathway. The use of molecularly targeted therapies has markedly improved the prognosis of relatively small subgroups of patients whose tumors harbor activated oncogenes, including EGFR mutations or ALK rearrangements, for whom a plethora of effective inhibitors are currently available. Recently, for NSCLC patients harboring the oncogenic BRAF V600E mutation, dabrafenib and trametinib combination has emerged as a novel, effective therapeutic option, thus suggesting that the clinical scenario for oncogene-addicted tumors will continue to evolve over the next years.

Despite all the therapeutic advances, the 5-year survival rate for advanced lung cancer patients remains unsatisfactory. It is widely recognized that a small proportion of patients do not respond to targeted agents although harboring activated oncogenes and others who undergo rapid disease progression although showing initial objective responses. Both cases of treatment failure can be partly explained by the fact that lung cancer has been shown to be a highly heterogeneous disease. Intratumor heterogeneity may present major challenges to personalized medicine, but its recognition and characterization in primary and metastatic solid cancers, both at baseline or when they become unresponsive to treatment, byhighly sensitive, large-scale sequencing techniques may offer novel therapeutic targets or treatment combinations and sequential strategies to improve patients outcomes. Liquid biopsy has been demonstrated as a viable surrogate to tissue biopsy for assessing tumor-specific genetic and epigenetic alterations in lung cancer. Liquid biopsy can potentially offer a wider representation of the entire molecular profile of both primary tumor and metastatic sites, thereby overcoming tissue genotyping limitations due to heterogeneity of metastatic tumors and offering the opportunity of tailoring treatment based on the cadre of mutations identified. In conclusion, liquid biopsies have several advantages: minimally invasively sampling and dynamically monitoring of tumor initiation and progression, with the potential of predicting prognosis and guiding therapeutic strategies. There still exist some challenges, such as the sensitivity/specificity, which but in most cases have been evaluated in studies with small sample size (few hundreds of patients), and should be confirmed in larger cohorts (thousands of patients). The possibility of rapidly repeating the analysis and improving technical limitations, as well as the planning of new analyses in large-scale clinical trials, is reassuring. Another concern is the inability to evaluate morphological transformations, such as transformation of NSCLC to small cell lung cancer, which is one of the known mechanisms of resistance to EGFR TKIs. In this case, liquid biopsies should be used as a strong supplement to tissue detection methods. Prospective studies with novel targeted agents are indeed ongoing with simultaneous collection of tissue and blood samples for correlative molecular analyses at baseline and at different points during the treatment and will probably provide new insights into molecular alterations underlying primary and acquired resistance.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work was funded by Dutch Cancer Society (KWF) grant (no. 10410), Cancer Center Amsterdam Foundation grants (no. 2012/2015) to E Giovannetti, and Associazione Italiana per la Ricerca sul Cancro Start-Up grant to E Giovannetti and N Funel.