Lung cancer burden is increasing, with 2 million deaths/year worldwide. Current limitations in early detection impede lung cancer diagnosis when the disease is still localized and thus more curable by surgery or multimodality treatment. Liquid biopsy is emerging as an important tool for lung cancer early detection and for monitoring therapy response. Here, we reviewed recent advances in liquid biopsy for early diagnosis of lung cancer. We summarized DNA- or RNA-based biomarkers, proteins, autoantibodies circulating in the blood, as well as circulating tumor cells (CTCs), and compared the most promising studies in terms of biomarkers prediction performance. While we observed an overall good performance for the proposed biomarkers, we noticed some critical aspects which may complicate the successful translation of these biomarkers into the clinical setting. We, therefore, proposed a roadmap for successful development of lung cancer biomarkers during the discovery, prioritization, and clinical validation phase. The integration of innovative minimally invasive biomarkers in screening programs is highly demanded to augment lung cancer early detection.

Background: Progression to stage IV disease remains the main cause of breast cancer-related deaths. Increasing knowledge on the hematogenous phase of metastasis is key for exploiting the entire window of opportunity to interfere with early dissemination and to achieve a more effective disease control. Recent evidence suggests that circulating tumor cells (CTCs) possess diverse adaptive mechanisms to survive in blood and eventually metastasize, encouraging research into CTC-directed therapies. Methods: On the hypothesis that the distinguishing molecular features of CTCs reveal useful information on metastasis biology and disease outcome, we compared the transcriptome of CTCs, primary tumors, lymph-node and lung metastases of the MDA-MB-231 xenograft model, and assessed the biological role of a panel of selected genes, by in vitro and in vivo functional assays, and their clinical significance in M0 and M+ breast cancer patients. Results: We found that hematogenous dissemination is governed by a transcriptional program and identified a CTC signature that includes 192 up-regulated genes, mainly related to cell plasticity and adaptation, and 282 down-regulated genes, involved in chromatin remodeling and transcription. Among genes up-regulated in CTCs, FADS3 was found to increases cell membrane fluidity and promote hematogenous diffusion and lung metastasis formation. TFF3 was observed to be associated with a subset of CTCs with epithelial-like features in the experimental model and in a cohort of 44 breast cancer patients, and to play a role in cell migration, invasion and blood-borne dissemination. The analysis of clinical samples with a panel of CTC-specific genes (ADPRHL1, ELF3, FCF1, TFF1 and TFF3) considerably improved CTC detection as compared with epithelial and tumor-associated markers both in M0 and stage IV patients, and CTC kinetics informed disease relapse in the neoadjuvant setting. Conclusions: Our findings provide evidence on the potential of a CTC-specific molecular profile as source of metastasis-relevant genes in breast cancer experimental models and in patients. Thanks to transcriptome analysis we generated a novel CTC signature in the MDA-MB-231 xenograft model, adding a new piece to the current knowledge on the key players that orchestrate tumor cell hematogenous dissemination and breast cancer metastasis, and expanding the list of CTC-related biomarkers for future validation studies.

For cancer management, predicting and monitoring response to treatment and disease progression longitudinally is crucial due to changes in tumor biology and therapy responsiveness over time. However, solid tumors are usually sampled only at time of initial diagnosis, as obtaining tissue biopsies is an invasive procedures with associated risks. Thus, there is a pressing need for approaches able to serially detect function-related reliable biomarkers reflecting treatment response and/or disease progression through easy noninvasive procedures, amenable for longitudinal analysis of tumor molecular features. Recent evidences indicate that blood and other body fluids could replace invasive surgical biopsies and represent a “liquid biopsy” containing cells and nucleic acids released by primary and metastatic lesions, reflecting their biological features and allowing identification of clinically useful biomarkers and treatment-induced cancer adaption processes. The development of new and highly sensitive technologies that allow to detect and characterize circulating tumor cells, to identify cell-free nucleic acids (circulating tumor-associated microRNAs and cancer-specific mutations in circulating DNA) and to measure their eventual dynamic changes represents therefore a major achievement for disease monitoring. However, notwithstanding preliminary findings support the prognostic and/or predictive role of this new generation of biomarkers, there are a number of technical and biological caveats that still require additional studies to demonstrate and validate their clinical utility. A unique opportunity to rapidly assess the contribution of circulating tumor cells and cell-free nucleic acids to patient management and to personalized medicine could derive by their combined consideration in the neoadjuvant setting.

Purpose: To compare circulating tumor cell (CTC) detection rates in patients with early (M₀) and metastatic (M₊) breast cancer using 2 positive-selection methods or size-based unbiased enrichment. Methods: Blood collected at baseline and at different times during treatment from M₀ patients undergoing neoadjuvant therapy and from M₊ women starting a new line of treatment was processed in parallel using AdnaTest EMT-1/ and EMT-2/Stem CellSelect/Detect kits or ScreenCell Cyto devices. CTC positivity was defined according to the suggested cutoffs and cytological parameters, respectively. Results: Higher CTC detection rates were obtained with the AdnaTest approach when using for CTC-enrichment antibodies against ERBB2 and EGFR in addition to MUC1 and the classical epithelial surface marker EPCAM (13% vs. 48%). In M₀ patients mainly, CTC positivity rates further increased when EMT- and stemness-related marker expression (PIK3CA, AKT2 and ALDH1) was evaluated in addition to EPCAM, MUC1 and ERBB2. When the physical properties of tumor cells were exploited, CTCs were detected at higher percentages than with positive-selection-based methods, without any difference between clinical stages (78% in M₀ vs. 72% in M₊ cases at baseline). Circulating tumor microemboli (CTMs) were detected in addition to single CTCs with significantly higher frequency in M₀ than M₊ samples (78% vs. 27%, p = 0.0002). Conclusions: Different approaches for CTC detection probably identify distinct tumor cell subpopulations, but need technical standardization before their clinical validity and biological specificity may be adequately investigated. The distinct role of CTMs compared with CTCs as prognostic and predictive biomarkers represents a further challenge.

Hematogenous dissemination may occur early in breast cancer (BC). Experimental models could clarify mechanisms, but in their development, the heterogeneity of this neoplasia must be considered. Here, we describe circulating tumor cells (CTCs) and the metastatic behavior of several BC cell lines in xenografts. MDA-MB-231, BT-474, MDA-MB-453 and MDA-MB-468 cells were injected at the orthotopic level in immunocompromised mice. CTCs were isolated using a size-based method and identified by cytomorphological criteria. Metastases were detected by COX IV immunohistochemistry. CTCs were detected in 90% of animals in each model. In MDA-MB-231, CTCs were observed after 5 weeks from the injection and step wisely increased at later time points. In animals injected with less aggressive cell lines, the load of single CTCs (mean ± SD CTCs/mL: 1.8 ± 1.3 in BT-474, 122.2 ± 278.5 in MDA-MB-453, 3.4 ± 2.5 in MDA-MB-468) and the frequency of CTC clusters (overall 38%) were lower compared to MDA-MB-231 (946.9 ± 2882.1; 73%). All models had lung metastases, MDA-MB-453 and MDA-MB-468 had ovarian foci too, whereas lymph nodal involvement was observed in MDA-MB-231 and MDA-MB-468 only. Interestingly, CTCs showed morphological heterogeneity and were rarely associated to host cells. Orthotopic xenograft of BC cell lines offers valid models of hematogenous dissemination and a possible experimental setting to study CTC-blood microenvironment interactions.