Treatment response and tumor evolution: lessons from an extended series of multianalyte liquid biopsies in a metastatic breast cancer patient.

Currently, clinical characterization of metastatic breast cancer is based on tissue samples taken at time of diagnosis. However, tissue biopsies are invasive and tumors are continuously evolving, which indicates the need for minimally invasive longitudinal assessment of the tumor. Blood-based liquid biopsies provide minimal invasive means for serial sampling over the course of treatment and the opportunity to adjust therapies based on molecular markers. Here, we aim to identify cellular changes that occur in breast cancer over the lifespan of an affected patient through single-cell proteomic and genomic analysis of longitudinally sampled solid and liquid biopsies. Three solid and 17 liquid biopsies from peripheral blood of an ER+/HER2- metastatic breast cancer patient collected over 4 years and eight treatment regimens were analyzed for morphology, protein expression, copy-number alterations, and single-nucleotide variations. Analysis of 563 single morphometrically similar circulating tumor cells (CTCs) and 13 cell-free DNA (cfDNA) samples along with biopsies of the primary and metastatic tumor revealed progressive genomic evolution away from the primary tumor profiles, along with changes in ER expression and the appearance of resistance mutations. Both the abundance and the genomic alterations of CTCs and cfDNA were highly correlated and consistent with genomic alterations in the tissue samples. We demonstrate that genomic evolution and acquisition of drug resistance can be detected in real time and at single-cell resolution through liquid biopsy analytes and highlight the utility of liquid biopsies to guide treatment decisions.

Preanalytical Variables for the Genomic Assessment of the Cellular and Acellular Fractions of the Liquid Biopsy in a Cohort of Breast Cancer Patients.

Liquid biopsy allows assessment of multiple analytes, providing temporal information with potential for improving understanding of cancer evolution and clinical management of patients. Although liquid biopsies are intensely investigated for prediction and response monitoring, preanalytic variables are of primary concern for clinical implementation, including categories of collection method and sample storage. Herein, an integrated high-density single-cell assay workflow for morphometric and genomic analysis of the liquid biopsy is used to characterize the effects of preanalytical variation and reproducibility of data from a breast cancer cohort. Following prior work quantifying performance of commonly used blood collection tubes, this study completes the analysis of four time points to assay (24, 48, 72, and 96 hours), demonstrating precision up to 48 hours after collection for assay sensitivity, highly reproducible rare cell enumeration, morphometric characterization, and high efficiency and capacity for single-cell genomic analysis. For the cell-free analysis, both freezing and use of fresh plasma produced similar quality and quantity of cell-free DNA for sequencing. The genomic analysis (copy number variation and single-nucleotide variation) described herein is broadly applicable to liquid biopsy platforms capable of isolating cell-free and cell-based DNA. Morphometric parameters and genomic signatures of individual circulating tumor cells were evaluated in relation to patient clinical response, providing preliminary evidence of clinical validity as a potential biomarker aiding clinical diagnostics or monitoring progression.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice.

Aberrant autophagy is a major risk factor for inflammatory diseases and cancer. However, the genetic basis and underlying mechanisms are less established. UVRAG is a tumor suppressor candidate involved in autophagy, which is truncated in cancers by a frameshift (FS) mutation and expressed as a shortened UVRAGFS. To investigate the role of UVRAGFS in vivo, we generated mutant mice that inducibly express UVRAGFS (iUVRAGFS). These mice are normal in basal autophagy but deficient in starvation- and LPS-induced autophagy by disruption of the UVRAG-autophagy complex. iUVRAGFS mice display increased inflammatory response in sepsis, intestinal colitis, and colitis-associated cancer development through NLRP3-inflammasome hyperactivation. Moreover, iUVRAGFS mice show enhanced spontaneous tumorigenesis related to age-related autophagy suppression, resultant ß-catenin stabilization, and centrosome amplification. Thus, UVRAG is a crucial autophagy regulator in vivo, and autophagy promotion may help prevent/treat inflammatory disease and cancer in susceptible individuals.

Herbicide resistance and biodiversity: agronomic and environmental aspects of genetically modified herbicide-resistant plants.

Farmland biodiversity is an important characteristic when assessing sustainability of agricultural practices and is of major international concern. Scientific data indicate that agricultural intensification and pesticide use are among the main drivers of biodiversity loss. The analysed data and experiences do not support statements that herbicide-resistant crops provide consistently better yields than conventional crops or reduce herbicide amounts. They rather show that the adoption of herbicide-resistant crops impacts agronomy, agricultural practice, and weed management and contributes to biodiversity loss in several ways: (i) many studies show that glyphosate-based herbicides, which were commonly regarded as less harmful, are toxic to a range of aquatic organisms and adversely affect the soil and intestinal microflora and plant disease resistance; the increased use of 2,4-D or dicamba, linked to new herbicide-resistant crops, causes special concerns. (ii) The adoption of herbicide-resistant crops has reduced crop rotation and favoured weed management that is solely based on the use of herbicides. (iii) Continuous herbicide resistance cropping and the intensive use of glyphosate over the last 20 years have led to the appearance of at least 34 glyphosate-resistant weed species worldwide. Although recommended for many years, farmers did not counter resistance development in weeds by integrated weed management, but continued to rely on herbicides as sole measure. Despite occurrence of widespread resistance in weeds to other herbicides, industry rather develops transgenic crops with additional herbicide resistance genes. (iv) Agricultural management based on broad-spectrum herbicides as in herbicide-resistant crops further decreases diversity and abundance of wild plants and impacts arthropod fauna and other farmland animals. Taken together, adverse impacts of herbicide-resistant crops on biodiversity, when widely adopted, should be expected and are indeed very hard to avoid. For that reason, and in order to comply with international agreements to protect and enhance biodiversity, agriculture needs to focus on practices that are more environmentally friendly, including an overall reduction in pesticide use. (Pesticides are used for agricultural as well non-agricultural purposes. Most commonly they are used as plant protection products and regarded as a synonym for it and so also in this text.).