Paper - Review

DOI: 10.1038/s41420-020-00373-0

Abstract

The sequencing technologies
→ allowed → the generation of (huge amount) ← of molecular data
← from a single cancer specimen
→ allowing → the clinical oncology → to enter the era of the precision medicine

❗: new details ← on cancer pathogenesis
❓: Relies ← on tissue biopsies
∵ Unable → to capture → the dynamic nature of cancer ← through its revolution

Blood
→ are analyzed applying the techniques
→ used for the generation of omics data

This approach
→ would allow → to take into account (tumor heterogeneity)
← in a time dependent manner
∴ In a somehow "real-time" ← understanding of (cancer evolution)

Background

Clinical oncology
→ rely on the increasing amount of molecular data
← which can be obtained ← from single cancer specimens

The molecular profiling (← of gene mutations)
→ 1⃣ for prognostic predictions 2⃣ for therapy selection
→ can be → a standard approach
← in many cancer entities since decades

Purely histopathologic cancer diagnosis
← to a molecular-based one
→ to develop more (accurate & complex) prognostic scores

∴ Opened → the era of targeted therapies
→ to precision medicine

Intelligent drugs
→ designed → to specifically target → a precise molecular objective
→ have entered → the clinics alongside classic chemotherapy

This new options
→ generated → great therapeutical advances
← in many cancer contexts
→ results in a time limited controlled status of the disease
← leading to cancer progression ← in most cases

This inevitable failure
∵ 1⃣ to the heterogeneous nature of cancer 2⃣ to the evolutive pressure ← that is mediated by cancer treatment itself

Tumor biopsies
→ can give → limited information ← on a single cancer entity
∵ They cannot account 1⃣ for its intrinsic tumor heterogeneity 2⃣ for its evolution during time
∵ their invasive nature ←which impairs the possibility of their repetition → to follow cancer evolution

Great advances (← in the sequencing technologies)
→ are at the roots of the generation (← of many more accurate molecular data)
← from single cancer specimens

Whole cancer genomics analysis
→ can now be performed ← at relatively low costs
e.g. whole transcriptomic & proteomic

These techniques
→ led to the generation of vast amounts of (data derived)
← from a single cancer specimen
∴ determining → a molecular deep characterization of a single cancer
← 1⃣ in a precise 2⃣ in limited time of the disease

Cancer specimens
→ are NOT ❌ → the most ideal source material
→ for capturing → the dynamic nature of cancers
∵ they cannot be repeated ← during the evolution of the disease
∵ their invasive nature

Blood
→ harbor many biological entities ← which directly derive from cancer itself
→ can be exploited ← as the ideal source ← for liquid biopsies

Liquid biopsies in cancer

The circulating blood
→ reflects 1⃣ primary 2⃣ metastatic tumor biology
∵ tumor cells → are more prone than normal cells
→ to release (nucleic acids) ← into the bloodstream upon death

ctDNA
← circulating tumor DNA
→ can be purified ← from the plasma of cancer patients
→ can be analyzed → through 1⃣ next generation sequencing 2⃣ targeted sequencing

Both 1⃣ normal 2⃣ tumoral DNA
→ are circulating
→ cannot ❌ be properly separated form each other
❗: The most abundant source of (cell-free DNA) → is the tumoral one

Liquid biopsies
→ can rely ← on other aspects of (cancer tumorigenesis)
e.g. 1⃣ CTCs ← circulating tumor cells 2⃣ ECVs ← extra-cellular vesicles

CTCs
→ can be isolated ← from peripheral blood
→ can be discriminated form normal cells
← using 1⃣ anti-EpCAM 2⃣ anti-CK 3⃣ CD45 selection methods

CTCs
→ correlate ← with 1⃣ prognosis 2⃣ a metastatic disease status 3⃣ their clinical role
← in defining cancer prognosis
→ has been stated ← by the FDA approval
→ for their detection ← in 1⃣ breast cancer 2⃣ prostate cancer

CTCs
→ account → for tumor heterogeneity
∵ the circulating cells → reflect cancer sub-clones
∵ the circulating cells → can be the ideal source ← for 1⃣ whole genome sequencing 2⃣ transcriptomic analysis

CTCs
→ can also be used → to establish (organoids cultures)
→ which might be → a valuable in vitro reproduction ← of an individual cancer

ECVs
→ can be separated ← from plasma
→ through different methodologies

ECVs
→ show → highly enriched (exosome proteins)
∴ ECVs → constitute (a valuable source) ← of information ← from a single cancer

Proteins
→ are not the only components of ECVs
∵ 1⃣ tumor DNA 2⃣ tumor RNA

Proteins
→ are NOT ❌ → solely inert (tumoral material)
∵ 1⃣ Proteins → can function ← as signal transductor 2⃣ Proteins → are highly present ← in the tumor micro-environment
∵ 1⃣ Proteins → can signal → through an (autocrine & paracrine) pathways 2⃣ Proteins → can be released in the bloodstream ← for long distance signaling

Blood
→ is surely → the most characterized tissue

The microbiota
→ creates a complex network
← can influence (the tumor micro-environment) ← in a very heterogeneous way
← relies on the intrinsically heterogeneity of the microbiome itself

The main advantage ( ←of a liquid biopsy approach)
→ stands with the possibility → to capture tumor heterogeneity
→ through its evolution
∵ They are NOT ❌ harmful → for the patients

Liquid biopsies for breast cancer

Breast cancer
→ represent → a striking evidence
→ for the advantage of liquid biopsies
← use in the everyday medical treatment

CTCs
→ arise ← during early stages of breast cancer
← on (triple negative breast cancer) (TBNC)
← this (cell population) ← at diagnosis → is quite heterogenous
← in terms of expression of (hormone receptors)

Deeper understanding
← on breast cancer CTCs
→ comes from (the evidence) ← that this cell population express
→ both 1⃣ epithelial ← e.g. 1⃣ keratins 2⃣ EpCAM 3⃣ cadherin1 2⃣ mesenchymal markers ← 1⃣ cadherin 2 2⃣ fibronectin 3⃣ serpine peptidase inhibitor
∴ They might be subject → to the epithelial-to-mesenchymal (EMT) reprogramming

The most represented subgroup (← of CTCs)
→ expresses mesenchymal markers
∴ EMT plays → a pivotal role ← in their generation

CTCs (← in breast cancer)
→ correlate ← with metastasis development
EMT → is highly associated
→ to the development of a metastatic disease
← from a clinical point of view

A liquid biopsy
→ does NOT ❌ only rely ← on the evaluation of CTCs

The secretion of proteins
← from tumor cells
→ has been investigated ← through label-free quantitative proteomics approaches
← on plasma samples

Tumoral secretomes
→ are highly abundant in plasma
∵ Cancer cells → are much more prone
→ to 1⃣ shedding 2⃣ releasing → their content into the bloodstream

Secreted proteins
← combined to (RNA sequencing) approaches
→ has been proven → to be able to distinguish
← among (breast cancer) phenotypes ←with different prognosis
→ has probed → to be a valuable strategy → for outcome prediction

Proteins
→ are NOT ❌ only → secreted ← in the bloodstream
Proteins
→ can also be incorporate ← within tumor-derived extra-cellular vesicles
→ together with other components ← e.g. 1⃣ DNA 2⃣ RNA

One (← of the components of tumor derived ECVs)
→ is miRNAs

Circulating miRNA
→ are highly heterogeneous compartment (← of the circulating RNA)
Circulating miRAN evaluation
→ is quite difficult ← in plasma
❗: they are enriched ← in ECVs

The other abundant tumor-derived circulating component
← derived from a liquid biopsy
→ is ctDNA

ctDNA
→ has been correlated → to patients' relapse ← after 1⃣ surgery 2⃣ adjuvant treatment
→ predicting ← before (the clinical evidence) ← of the metastatic disease
→ also correlating ← with the disease burden

ctDNA
→ correlate → to tumor dynamics
← in breast cancer patients ← undergoing 1⃣ surgical 2⃣ medical therapy

Some methodological limitations
→ are still embedded
← in the liquid biopsies

ctDNA sequencing
→ relies ← on small circulating fragments
← which are diluted ← among the normal circulating DNA
∴ ctDNA → represent an impure source of information

Liquid biopsies in neuroblastoma

NB
← neuroblastoma
→ accounts → a significant fraction (10%) ← of death ← from children malignancies

Genomic abnormalities
→ have indeed been → sufficiently 1⃣ defined 2⃣ linked
→ to the clinical presentation

Neuronal differentiation markers
→ have been associated → to the pathogenesis of the disease

Diagnostic procedures → for NM
→ rely on → 1⃣ radiographic body imaging 2⃣ nuclear medicine examination 3⃣ evaluation of serum levels ← of neuron-specific enzymes 4⃣ urinary levels ← of vanillylmandelic acid (VMA) & homovanillic acid (HVA)

❗: Sensitivity (← of these techniques) → is low
→ to represent → proper early detection methods
∴ Their diagnostic value
→ emerges → quite late
←when the cancer has 1⃣ developed into clinical stage 2⃣ discrete lumps evident

Detection of cell-free DNA (cfDNA) → for NB
→ could improve → 1⃣ effectiveness ← of diagnostic methods 2⃣ tolerability ← of medical procedure

Those early data
→ indicated that → MYCN DNA (← in the blood circulation) → is an early process in disease
∴ Those early data → offers → a potential novel marker → for patient follow-up after treatment

Subsequent studies
→ have reduced → the optimism

ALK
→ is a tyrosin kinase receptor
← subjected to (copy number amplification) ← in NB cases

∴ Detection of ALK circulating DNA
→ has been explored → to achieve approaches
→ 1⃣ to monitor relapse 2⃣ to predict drug resistance

Good concordance
← between (primary tumor) 🆚 (liquid biopsies)
→ has been revealed ← in detection of several chromosomal variations

Conclusion

Oncology
→ has entered → the precision medicine era
← with the technological ability → to characterized (a single cancer) ← from multi-omics approaches

This huge amount ←of biologic details
→ does NOT ❌ (readily & dynamically) translate
← into the clinical practice
∵ it is usually performed ← on the cancer tissue itself

The improvement of (our understanding ← of cancer biology)
← with the discovery ← that tumoral material ← which circulates in the bloodstream
→ has give the opportunity → to conceive
→ the idea that blood itself → might represent a cancer biopsy per se

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