Microfluidics and Lab-on-a-Chip for biomedical applicationsChapter 6 : Cancer diagnostics & monitoring.

By Stanislas

CNRSUniversité de Lyon, FRANCEStansan International Group

CONTENT

Chapter 1: Introduction.

Chapter 2 : Basic principles of Microfluidics.

Chapter 3 : Basis of molecular biology and analytical tools.

Chapter 4 : Micromanufacturing.

Chapter 5 : Lab-on-a-Chip & applications.

Chapter 6 : Cancer diagnostics and monitoring.

A sinister aspect of the changes in a cell are the transformations that occur with accumulated genetic mistakes. Critical "gateway genes" that control fundamental metabolic and control pathways become mutated and the cell becomes unstable and starts unregulated growth. This is a highly complex process which is called CANCER.

Most cancer deaths are not caused by the growth of the primary tumor, but result from its invasive spread to secondary sites and the subsequent formation of metastasis.

ABOUT CANCER

2.9 million new cases of cancer and more than 1.7 million cancer deaths in Europe in 2004 (WHO report)

lung cancer was the commonest form of cancer diagnosed (13.2%) and of cancer death (20%)

colorectal cancer was almost equally common (13%), it represented a smaller proportion of deaths (11.9%).

among women, breast cancer was by far the most common, representing 27.4% of all female cases and it was also the biggest killer with nearly 130,000 deaths – 17.4% of the total.

the ageing of the European population will cause these numbers to continue to increases by +50% in 2020.

it's mandatory to introduce completely new technologies for early diagnosis, therapy and follow up of the cancer, which become a major public health problem in both industrialised and in developing countries.

ABOUT CANCER

A cancer marker can be defined as a molecule, a process or a substance, which is altered quantitatively or qualitatively in pre-cancerous or cancerous conditions.

What are cancer markers ?

Thus, cancer markers can be a specific DNA mutation, mRNA, protein or process (apoptosis, angiogenesis, proliferation, etc.) measured by an appropriate assay.

The types of specimen in which cancer markers can be detected may be of various nature: tissue, blood (plasma/serum), saliva, urine…

Simple and nearly-non-invasive diagnostics/ & monitoring

DNA chips have been extensively used for research applications in academia and in industrial laboratories.

A big progress in bioinformatics is still needed in order to be able to explore in a reliable way the DNA data in the field of cancer.

It is expected that many DNA chip for cancer diagnostic and monitoring will be developed in the next few years.

One of challenges for the Biochip industry is also the integration of microarrays with microfluidics, in order to achieve Microsystems, which include the extraction of the genetic material (e.g. DNA or RNA from white blood cells or from rare circulating tumor cells), purification and amplification of the extracted material and, finally, the analysis of this material with DNA chips.

MOLECULAR DIAGNOSTICS

diagnostic cancer marker is a marker that will aid in detection of malignant disease in a patient; a diagnostic marker should exhibit both high levels of diagnostic sensitivity and specificity;

prognostic marker gives the clinician a tool for estimating the risk of disease recurrence and/or cancer-related death;

predictive cancer marker will foretell how the patient is going to respond to a given therapy;

monitoring markers are used during follow-up of patients who do or do not receive anti-cancer therapy ; detection of recurrence or remission.

Categories of markers and they clinical utility

Basic Diagnostic Test Interpretation

Disease present Disease absentTest positive True positives (TP) False positives (FP)Test negative False negative (FN) True negatives (TN)

Sensitivity is the proportion of patients with disease who test positive. SE = TP / (TP+FN)“False Negative Ratio” : FN / (FN + TP) = 1 - SE

Specificity is the proportion of patients without disease who test negative. SP = TN / (TN + FP) >>> FP = TN / (1/SP - 1)“False Positive Ratio” : FP / (TN + FP) = 1 - SP

Predictive value of a positive test is the proportion of patients with positive tests who have disease. It measures how well the test rules in disease. PVP = TP / (TP+FP)

Predictive value of a negative test is the proportion of patients with negative tests who do not have disease. It measures how well the test rules out disease. PVN = TN / (TN+FN)

back

IN REVIEWING LABORATORY TEST RESULTS:

No single test is 100% accurate for specificity, sensitivity or predictive value.

Any particular laboratory result may be inaccurate, for a variety of reasons.

The degree of abnormality and the significance of the abnormality are proportional. That is, marginal variations likely have less significance than wide variations.

Multiple test abnormalities are more likely significant that single test abnormalities.

Test results, when possible, should be compared over time, preferably with results from the same laboratory. When possible, comparison with the patient’s test results prior to illness may be helpful.

Le progrès rapide de la Génomique et de la Protéomique conduit à la compréhension des bases moléculaires des cancers et à la découvertes des nouveaux marqueurs.

Il est important de suivre la concentration de ces marqueurs.

La technologie Lab-on-a-Chip permettra le développement de méthodes d’analyse rapides, robustes et fiables pour les cabinets de médecin.  

Chronology of genetic and molecular lesions.

Normal Hyperplasia Métaplasia Dysplasia ISC Carcinoma

UPA-ST3+(Epith)

3p-9p-

Aneuploidy

17p-

P53mut

Rasmut

Protéases uPA, ST3(Strom a)

Télom erase +

Rb-FHIT

Exemple du suivi d’un marqueur cancéreux au cours d’une chimiothérapie

Graphique de la chute d’un marqueur (HCG) au cours d’une chimiothérapie d’une tumeur testiculaire.

Si un traitement est efficace, on observe une baisse de la concentration de marqueur.

Récession de la Récession de la tumeurtumeur

Le grand intérêt des analyseurs proposés réside dans le fait que le patient pourra bénéficier d'analyses beaucoup plus fréquentes permettant de détecter à temps une possible évolution néfaste.

A high level of HCG in the blood indicates that a cancer of the placenta called gestational trophoblastic neoplasia (GTN) may be present. This cancer continues to produce HCG. Some testicular and ovarian cancers resemble GTN because they both arise from reproductive cells called germ cells. These cancers also make HCG and this marker is used in their diagnosis and in monitoring their response to therapy. In the example below, note almost day-by-day changes in the concentration of the HCG marker.

Unfortunately, using standard diagnostic techniques, nobody today can be followed-up so closely. An enormous advantage of the instruments which could be developed using Lab-on-a-Chip

Thanks to Lab-on-a-Chip, patients could be monitored frequently, which will allow to adjust at time the treatment or, after the treatment, to detect at time possible recurrent cancer. However, up to now there is no such devices.

Need of frequent testing

Modern oncology must be based on systematic and frequent quantitative analysis of tumor markers which can :

help in diagnosis

be used as indicators of the clinical response of the cancer to various treatment modalities, in order to personalise or to adjust at time this treatment.

after the treatment, the patient must be followed up very closely in order to detect at time a possible recurrent cancer.

Unfortunately, using standard analytical techniques, nobody today can be followed-up as closely as necessary.

Full names of

markers cited

in the Table1

Details :

http://www.spendloveresearch.org/ResearchPages/tumor&cancertypes.htm

Example of Cancer Markers

ApproachSeparation of bio-molecules (proteins and/or DNA fragments) by electro-chromatography carried out in multiple microfluidic channels; this separation is coupled with nucleic acid hybridization reaction (DNA) or immunological reactions (proteins) in liquid phase or using appropriate ligands bound to the separation matrix.

Integration of new nano-structured materials into the microfluidic channels for micro/nano filtering or as new type of matrix for Improved separation techniques of bio-molecules - porous Si and nano-structured polymers.

Optical integration in the Lab-on-a-Chip, which will allow a dramatic reduction of the dimension and price of the control unit.

Integrated optics in the Lab-on-a-Chip for the redistribution of the excitation light and collection of the fluorescence signal - spectacular improvement of the performances, multiple separation columns…

heterogeneous integration of various materials (Silicon, glass, polymers) combining various functions : integrated optics, integrated microelectronics, microfluidics - micro-nano components for advanced biological functions etc..

packaging issues for future bath fabrications of such devices on large heterogeneous substrates involving silicon/glass/plastic wafers.

Operation with a drop of blood

USB

USB

The complete system will make use of disposable chip sensors with a size of a credit card and a control unit with a size of a book.

The developed instruments will be autonomous and simple and compatible with straightforward use (drop of blood). The developed instruments must have sufficient autonomy and simplicity for point-of-care operation (e.g. in physician cabinets).

Finger print protected smart card for data storage

Power supply

“heart of the system”

Cancer Prevention and Monitoring Card

Principe 1 :

Détermination de la concentration des marqueurs cancéreux, qui seront des protéines marqueurs, présents dans un liquide physiologique facilement accessible (sang, sérum…).

Approche 1 :

Séparation des diverses protéines de la solution par électro--chromatographie dans un système microfluidique Lab-on-a-Chip multicanal couplé à des réactions immunologiques spécifiques (protéines).

Principe 2 :

Intégration des Bio-puces à l’intérieur des canaux micro-fluidique.

Variantes technologiques de la réalisation.

Bio-puces situées à l’intersection d’un canal micro- fluidique avec un guide optique intégré dans les Lab-on-a-Chip.

Calibration-Optique- Micro-fluidique- Biologique

Approche 2 :

Détection de marqueurs biologiques, tels que ;-ADN, protéines….dans le domaine de la santé, défense, agro-alimentaire, contrôle d’environnement…

Dispositif micro-fluidique :-miniaturisation, très petite quantité de la solution à analyser…-sensibilité accrue

Intégration : optique + micro-fluidique :-miniaturisation du système extérieur de contrôle-sensibilité accrue.

Détection multiparamétrique :-meilleure interprétation des résultats.

-Point important :Point important : intégration Bio-puce & Lab-on-a-Chip

The detection of protein cancer markers will be obtained here by the integration on the Chip of the following blocks :

The most general block diagram of the Chip for the detection of molecular cancer markers is the following :

Functional Blocks

Silicon Micro-Needle

Minimally-Invasive Systemsfor blood sampling and drug

delivery

Examples of plasma extraction techniques

Enrichment and purification of low abundance proteins

Par example by affinity chromatography :

Example of a simple geometry of the electrophoretic separation

coupled with immunoreaction

CONTROL UNIT

Conception de l'architecture générale et choix des composants de l'unité de contrôle.

Développement de la première maquette d'un analyseur composé d’un capteur à utilisation unique interchangeable (comportant le Lab-on-a-Chip) et d'une unité de contrôle de la dimension approximative d'un livre.

Schéma général de l’unité de contrôle.

CONTROL UNIT

Photo de l’unité de contrôle en test. Exemple d’acquisition réalisée au laboratoire.

Notons que le développement de l’unité de contrôle entre parfaitement dans la thématique d’enseignement d’électronique à l’Ecole Centrale de Lyon. Nous avons pu ainsi intégrer la participation des élèves-ingénieurs dans ce développement dans les cadres des « Projets d’Etudes ».

CONTROL UNIT

Simplified Example of 3D integration of various materials and functions

Polymer « service » stage which contains electrical and vacuum interconnections with the control unit, related micro-channels, reservoirs for chemical products and waste.

Glass stage for electr-chromatographic separation of bio-molecules containing integrated optics and nano-structured materials in the separation channel.

Silicon stage for PCR, dielectrophoresis, integrated photodetectors, integrated electronic components (memory, DSP…)

Polymer stage which contains micro/nano filters (serum extraction), interface to the external word (sample and chemical inlets) ; it plays also a role of the packaging layer.

Medical CenterHome of a patient, of a doctor or a small analytical Lab

Alternative telecommunication technologies (terrestrial, wireless & mobile, satellite communications...)

Ensure integration with existing healthcare information infrastructure (HII) and consider compatibility with next generation clinical information systems

Protocols, security, encryption, authentication, privacy, etc...

Consider application requirements, limitations and tradeoffs

All the best for the future

THANK YOU FOR YOUR ATTENTION                                                                                                                                     

Any question ?