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Metachronous (MBC) and synchronous bilateral breast tumors (SBC) are mostly distinct primaries, whereas paired primaries and their local recurrences (LRC) share a common origin. Intra-pair gene expression variability in MBC, SBC, and LRC derives from time/tumor microenvironment-related and tumor genetic background-related factors and pairs represents an ideal model for trying to dissect tumor-related from microenvironment-related variability. Pairs of tumors derived from women with SBC (n = 18), MBC (n = 11), and LRC (n = 10) undergoing local-regional treatment were profiled for gene expression; similarity between pairs was measured using an intraclass correlation coefficient (ICC) computed for each gene and compared using analysis of variance (ANOVA). When considering biologically unselected genes, the highest correlations were found for primaries and paired LRC, and the lowest for MBC pairs. By instead limiting the analysis to the breast cancer intrinsic genes, correlations between primaries and paired LRC were enhanced, while lower similarities were observed for SBC and MBC. Focusing on stromal-related genes, the ICC values decreased for MBC and were significantly different from SBC. These findings indicate that it is possible to dissect intra-pair gene expression variability into components that are associated with genetic origin or with time and microenvironment by using specific gene subsets. [ABSTRACT FROM AUTHOR]

Background: The tumor microenvironment participates in the regulation of tumor progression and influences treatment sensitivity. In breast cancer, it also may play a role in determining the fate of non-invasive lesions such as ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive diseases, which is aggressively treated despite its indolent nature in many patients since no biomarkers are available to predict the progression of DCIS to invasive disease. In vitro models of stromal activation by breast tumor cells might provide clues as to specific stromal genes crucial for the transition from DCIS to invasive disease. Methods: normal human dermal fibroblasts (NHDF) were treated under serum-free conditions with cell culture media conditioned by breast cancer cell lines (SkBr3, MDA-MB-468, T47D) for 72 h and subjected to gene expression profiling with Illumina platform. Results: TGM2, coding for a tissue transglutaminase, was identified as candidate gene for stromal activation. In public transcriptomic datasets of invasive breast tumors TGM2 expression proved to provide prognostic information. Conversely, its role as an early biosensor of tumor invasiveness needs to be further investigated by in situ analyses. Conclusion: Stromal TGM2 might probably be associated with precancerous evolution at earlier stages compared to DCIS. [ABSTRACT FROM AUTHOR]

Simple Summary: Metastases cause the majority of breast cancer-related deaths. Circulating tumor cells (CTCs), and in particular CTC-clusters, are considered the seeds of metastasis, but their analysis in the early-stages of the disease has so far been limited by the fact that, by using conventional and epithelial-based technologies (as the FDA-approved CellSearch platform), they are more often detected in the metastatic setting. It is known, however, that cancer cells are heterogeneous and can downregulate the expression of epithelial markers, thus limiting the detection capability of epithelial-based technologies. Here, we show that it is possible to increase CTC-cluster detection by using an epithope-independent technology based on blood filtration, and in particular that this strategy allows to detect a high number of CTC-clusters in stage II-III breast cancer patients, before and during neoadjuvant treatment. Our results therefore offer a new opportunity to deepen our understanding of the cancer dissemination process in its early steps. The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies: CellSearch®, CellSieve™ filters, and ScreenCell® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria). At baseline, CTC-clusters were detected in 70% of EBC cases and in 20% of MBC patients (median number = 2, range 0–20, versus 0, range 0–15, p = 0.0015). Marker-independent approaches for CTC-cluster assessment improve detection and show that CTC-clusters are more frequent in EBC than in MBC patients, a novel finding suggesting that dissemination of CTC-clusters is an early event in BC natural history. [ABSTRACT FROM AUTHOR]

Simple Summary: Distant metastases derive from the shedding and dissemination of single cancer cells (CTCs) or circulating tumor emboli (CTMs) into circulation. Previous studies on CTMs were mainly run in patients with metastatic disease; however, we observed that CTMs are more frequently detected in patients with early-stage breast cancer. Here, we collected single CTMs and their relative primary tumor tissue samples in early-stage patients. By studying genomic aberrations, present in tumors cells and absent in normal cells, we predicted the tumor fraction thanks to a statistical model developed from a calibration curve with breast cancer cell lines. The tumor fraction ranged from 8% to 48% and CTMs contained specific and shared alterations with respect to tissue. Thus, CTMs may derive from different regions of the primary tumor or from occult micrometastases. Moreover, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be further explored in follow-up and mechanistic studies. Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood samples serially collected from 10 patients with metastatic RCC entering the TARIBO trial, we overcame this limitation using the marker-independent Parsortix™ approach for CTC-enrichment coupled with positive and negative selection with the DEPArray™ with single cell recovery and analysis for copy number alterations (CNA) by next generation sequencing NGS. Two CTC subpopulations were identified: epithelial CTC (eCTC) and non-conventional CTC (ncCTC) lacking epithelial and leukocyte markers. With a threshold ≥1CTC/10 mL of blood, the positivity rates were 28% for eCTC, 62% for ncCTCs, and 71% considering both CTC types. In two patients with detectable eCTCs at baseline, progression free survival was less than 5 months. In an index case, hierarchical structure by translational oncology (TRONCO) identified three clones among 14 CTCs collected at progression and at baseline, each containing cells with a 9p21.3loss, a well-known metastasis driving subclonal alteration. CTCs detection in RCC can be increased by marker-independent approaches, and CTC molecular characterization can allow detection of subclonal events possibly related to tumor progression. [ABSTRACT FROM AUTHOR]

Liquid biopsy (LB) is a non-invasive approach representing a promising tool for new precision medicine strategies for cancer treatment. However, a comprehensive analysis of its reliability for pancreatic cancer (PC) is lacking. To this aim, we performed the first meta-analysis on this topic. We calculated the pooled sensitivity, specificity, positive (LR+) and negative (LR−) likelihood ratio, and diagnostic odds ratio (DOR). A summary receiver operating characteristic curve (SROC) and area under curve (AUC) were used to evaluate the overall accuracy. We finally assessed the concordance rate of all mutations detected by multi-genes panels. Fourteen eligible studies involving 369 patients were included. The overall pooled sensitivity and specificity were 0.70 and 0.86, respectively. The LR+ was 3.85, the LR- was 0.34 and DOR was 15.84. The SROC curve with an AUC of 0.88 indicated a relatively high accuracy of LB for molecular characterization of PC. The concordance rate of all mutations detected by multi-genes panels was 31.9%. LB can serve as surrogate for tissue in the molecular profiling of PC, because of its relatively high sensitivity, specificity and accuracy. It represents a unique opportunity to be further explored towards its introduction in clinical practice and for developing new precision medicine approaches against PC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Enzalutamide and abiraterone have been shown to improve progression-free survival (PFS) and overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) patients. Moreover, some patients may not benefit from the inhibition of androgen receptor (AR) activity or, alternatively, may develop secondary resistance. Detection in patients' circulating tumor cells (CTCs) of ARV7, a splicing variant of AR lacking the ligand-binding domain, showed a link with treatment failure. Independent confirmation of the predictive role of CTC status combined with ARV7 detection is, therefore, a priority for extending personalized biomarker-driven treatments to all patients. In this prospective observational study, CTC status and the expression of AR and ARV7 were measured in 37 mCRPC patients, before starting treatment with enzalutamide or abiraterone, by employing commercially available kits. CTC status was positive in 21/37 patients: 46% and 24% of CTC-positive patients were defined as AR- and ARV7-positive, respectively. Kaplan–Meier estimates showed that positivity for each variable was significantly associated with poorer radiological PFS, PSA-PFS, and OS. All considered treatment outcomes worsened when going from CTC-negative to CTC-positive/ARV7-negative to CTC-positive/ARV7-positive patients, both in the global case series and in patients stratified into three groups based on basal PSA levels. Presently, technical approaches appear to be mature for introducing CTC/ARV7 tests in clinical practice. [ABSTRACT FROM AUTHOR]