WO2015018750A1

WO2015018750A1 - Sample separation using photoresist

Info

Publication number: WO2015018750A1
Application number: PCT/EP2014/066576
Authority: WO
Inventors: Reinhold Wimberger-Friedl; Danielle Elisa Willemine Clout; Christianne Rossette Maria DE WITZ
Original assignee: Koninklijke Philips N.V.
Priority date: 2013-08-09
Filing date: 2014-08-01
Publication date: 2015-02-12

Abstract

The invention relates to a method and a sample separation device (400) for separating material from a sample-ROI (region-of-interest) in a biological sample (11). The sample (11) is coated with a photoresist (20), which is then structured by irradiation to be soluble above the sample-ROI and insoluble elsewhere. By application of an appropriate solvent (431), the photoresist (20) is opened at the sample-ROI and the sample material below is extracted in one step. The extracted sample material is preferably subjected to molecular diagnostics (MDx). The solvent (431) may for example comprise a mixture of a base, a buffer, and a biological agent.

Description

Sample separation using photoresist

FIELD OF THE INVENTION

The invention relates to a sample separation device and a method for the separation of material from a region of interest in a biological sample. Moreover, it relates to a solvent that can be used in such a method.

BACKGROUND OF THE INVENTION

The US 2011/0177518 Al discloses a method for selective isolation of cellular material, said method comprising depositing a photosensitive material on the cellular material, exposing the photosensitive material through a photomask to light, and applying a developer to the photosensitive material in order to define an access well corresponding to a region of interest of the cellular material.

SUMMARY OF THE INVENTION

It would be advantageous to have means that allow for a facilitated processing of biological sample material of dedicated regions of interest.

This concern is addressed by a method according to claim 1 , a sample separation device according to claim 2, and a solvent according to claim 14. Preferred embodiments are disclosed in the dependent claims.

According to a first aspect, an embodiment of the invention relates to a method for the separation of material from a region of interest in a biological sample. Said region of interest in the sample will in the following be called "sample-ROI". It may for example correspond to one or more cells or cell fractions with specific features such as a particular color in a staining assay, wherein it is desirable to extract just the material of the sample-ROI for further testing. The method comprises the following consecutive steps:

a) Coating the sample at least partially with a layer of photoresist.

b) Irradiating said layer of photoresist selectively such that the photoresist is, after irradiation, soluble at (above) the sample-ROI but insoluble elsewhere. c) Applying a solvent to the photoresist that dissolves the soluble photoresist and that extracts sample material below (i.e. previously covered by) the dissolved photoresist.

The term "photoresist" shall denote any photosensitive material that changes its solubility with respect to an associated solvent (or class of solvents) when being exposed to electromagnetic radiation of an appropriate spectrum. The appropriate spectrum may for example comprise visible light, particularly of high photon energy, and/or UV light. The terms "soluble", "insoluble", "solubility" etc. are always defined with respect to a given solvent (the one used in step c).

The irradiation in step b) is assumed to take place with an intensity and a spectrum that are appropriate to change the solubility of the photoresist. The selectivity of the irradiation shall be spatial, meaning that certain areas of the photoresist are irradiated to induce solubility changes while others are not. Depending on the type of photoresist, the exposure to radiation may increase solubility ("positive photoresist") or decrease it ("negative photoresist"), allowing to induce a local change from insoluble to soluble or vice versa. As the photoresist will often be in a liquid state during the coating step a), some hardening or curing of the photoresist will typically occur between the coating and irradiation steps a) and b).

According to a second aspect, an embodiment of the invention relates to a sample separation device for the separation of material from a region of interest, called "sample-ROI", in a biological sample, said device comprising the following components:

A "coating unit" for coating the sample at least partially with a layer of a photoresist.

An "irradiation unit" for irradiating said layer of photoresist selectively such that the photoresist is, after irradiation, soluble above the sample-ROI and insoluble elsewhere.

An "extraction unit" for applying a solvent such that the soluble photoresist is dissolved and the sample material below is extracted.

The sample separation device provides equipment with which the method defined above can be executed. Explanations provided for the method are therefore analogously valid for the sample separation device and vice versa.

The method and the sample separation device have the advantage that they allow simultaneously for a removal of protective photoresist above a sample-ROI and an extraction of the biological material in said sample-ROI. This is achieved by structuring a layer of photoresist and then using an appropriate solvent that both opens the photoresist and extracts biological material at the sample-ROI.

A solvent for usage in the above method or with the above sample separation device is comprised by the invention, too. In the following, various preferred embodiments of the invention will be described in more detail that relate to the method, the sample separation device, and the solvent defined above.

Depending on the chemical composition of the applied photoresist, the biological sample at hand, and the intended further processing with the extracted material, different solvents will be suited for realizing the method and the sample separation device. The solvents will usually be mixtures of several chemical substances that serve different purposes in the whole process.

In a preferred embodiment, the used solvent comprises at least one (particularly at least two or three) of the following substances:

A base such as NaOH, and/or KOH.

- A buffer such as Tris-HCl.

A detergent such as sodium dodecyl sulfate (SDS) and/or

polyoxyethylene(20)sorbitan monolaurate (Tween 20).

A component such as beta-mercaptoethanol and/or dithiothreitol

(DTT).

- A biological agent, particularly an enzyme such as a proteinase K.

A chaotropic salt such as guanidinium isothiocyanate/hydrochloride. A solvent comprising a base is particularly suited in combination with a positive photoresist. Moreover, a base may induce lysis of cells of the sample material, thus helping to expose internal cellular material of interest. The base, e.g. NaOH, may preferably be present in the solvent in a concentration ranging between about 0.002 mol/1 to

about 0.30 mol/1, preferably between about 0.010 mol/1 to about 0.30 mol/1 or between about 0.002 mol/1 and about 0.1 mol/1 .

The buffer may particularly be adapted to stabilize a pH value of about 6 to about 9. This helps to prevent damaging of the biological sample material.

The biological agents are typically chosen for digesting proteins that are attached to the nucleic acids, like mRNA to accelerate their solubilization in the buffer. A proteinase may for example be used. In the further sample preparation protocol for PCR and/or sequencing such proteinases are denatured to avoid interference with the active enzymes of those MDx reactions. In general, any photoresist can be used provided that it is compatible with the biological sample material at hand. For example, the photoresists that are applied in microelectronic manufacturing can be used. Applicable photoresists may particularly be selected from the group consisting of epoxies, acrylates, methacrylates, SU-8,

poly(methylmethacrylate) (PMMA), poly(methylglutarimide) (PMGI), and phenol formaldehyde resin.

The selective irradiation of the photoresist layer may be achieved with the help of a mask representing the shape of the sample-ROI. The mask may be open in the sample-ROI and block irradiation outside, or vice versa. Preferably, selective irradiation is however achieved in a maskless manner, for example using selective guidance of a light beam by a mirror and/or lens. In this approach, a light beam may be directed only to areas where irradiation is desired, or a light beam may be scanned over the whole surface of the photoresist but be switched off or interrupted at locations that shall not be irradiated. In a preferred embodiment direct imaging is used (also called DLP of Texas Instruments) in which a mirror array is used to create the desired exposure pattern in one step from a collimated light beam. Maskless irradiation has the advantage that no elaborate production of a mask is required but that availability of (digital) data defining the sample-ROI is sufficient.

Sample material extracted from the sample-ROI may preferably be subjected to a molecular assay. In this context, the term "molecular assay" is to be understood in a broad sense, comprising any examination, test, or experiment, by which one or more parameters of the material from the sample-ROI that depend on its chemical composition may be determined. As an example, the molecular assay may comprise the (qualitative or quantitative) detection of particular proteins or nucleic acid sequences (e.g. tumor markers). In particular, the molecular assay may comprise PCR (e.g. q-PCR, qRT-PCR, RT-PCR, qrt-PCR, or digital PCR), sequencing (particularly next gen sequencing), or micro-array hybridization, or another molecular assay technology or a combination of these.

The biological sample will preferably be provided on some kind of carrier such that it can easily be handled. The carrier may particularly be a (microscope-) slide because this is readily available, well standardized to be compatible with many apparatuses, and of course suited for a microscopic inspection of the sample at hand.

According to a further embodiment, a cover may be disposed at (above) the sample-ROI, particularly during the extraction of the sample material. Thus a more or less closed chamber comprising the sample-ROI can be created. The cover will typically cover not only the sample-ROI but the whole sample. It may particularly be provided with an inlet for guiding the solvent to the sample-ROI and/or with an outlet for draining extracted sample material from the sample-ROI. A simple realization of the cover can be achieved with a cover slip as used in microscopy. A more versatile solution may be achieved with a dedicated cover having e.g. an adhesive to create a chamber of a defined volume that facilitates solvent introduction and removal.

In still another embodiment, an actuator may be provided for applying energy to the sample, particularly in the form of electromagnetic radiation, heat and/or ultrasound. The energy may for example be applied during the extraction of sample material from the sample-ROI. Thus the detachment of the desired sample material from the sample and/or a substrate (carrier) can be assisted. The actuator may optionally comprise a heater, a light source, and/or an ultrasonic transducer.

In general, the sample-ROI may be selected and defined by any procedure suited for the application at hand. In a preferred embodiment, the sample-ROI is derived from a second region of interest in an image of an object related to the sample. Said second region of interest in the image will in the following be called "image-ROI" to distinguish it from the sample-ROI. The "object related to the sample" may be the sample itself. It may however also be an additional (sub-) sample generated from the same material ("higher- level sample") as the sample at hand was taken from. It may for instance be a (preferably neighboring) slice generated from the same biopsy as the sample. The image may preferably be a microscopic image, for example generated with a digital scanning microscope. Moreover, the definition of the image-ROI may be done manually, e.g. by a pathologist, or automatically by image analysis software, or by a combination of both.

The aforementioned derivation of the sample-ROI from the selected image-ROI and/or the selection of the image-ROI are preferably executed while taking the capabilities of the photoresist patterning into account. Taking these limits into account will avoid the usage of a sample-ROI or an image-ROI that cannot be realized by the actual patterning procedure.

A given image-ROI may optionally be used to derive more than one sample-ROI. If for example several individual samples are obtained from a single "higher- level sample", for example several slices from a given biopsy, it will usually be necessary to identify the sample-ROI on each individual sample/slice. The image-ROI may then be identical, based on a single reference, however, the position of the sample-ROI and also its shape can differ for each individual slice. Hence there may be several sample-ROIs referring to the same image ROI for the purpose of separating material from consecutive slices originating from the same tissue block. It should be noted that the different sample-ROIs will usually require the individual patterning of a plurality of associated photoresist layers.

In another embodiment of the invention, an image of the sample may be generated after extraction of sample material from the sample-ROI. Such an image may be useful for verifying if the desired material has actually been extracted or not.

In still another embodiment, an image of the sample may be generated before the application of the photoresist to verify the position and/or deformation of the sample on the slide for an accurate later irradiation (patterning) of the photoresist. The sample material may be stained for instance with hematoxylin to provide morphological contrast for the registration of the image-ROI. Optionally an image can be taken after irradiation of the photoresist before the sample material is removed.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

In the drawings:

Fig. 1 schematically shows the extraction of sample material according to an embodiment of the present invention;

Fig. 2 schematically illustrates the step of coating sample material with a photoresist;

Fig. 3 schematically illustrates the step of patterning the photoresist by irradiation;

Fig. 4 illustrates the extraction of sample material from a sample-ROI in a cover;

Fig. 5 shows experimental results for two biological tests executed with sample material that was extracted in five different manners.

Like reference numbers refer in the Figures to identical or similar components.

DETAILED DESCRIPTION OF EMBODIMENTS

Pathology diagnostic investigation of patient material (e.g. tissue and cells) is the basis of many treatment decisions, in particular in oncology. Standard, thin slices from a biopsy are presented on microscope slides and stained according to certain protocols to visualize the morphology of the tissue, e.g. by Hematoxylin-Eosin (short H&E). More recently in situ staining for disease-specific biomarkers is being developed for companion diagnostics of targeted drugs, based on the specific binding of antibodies to proteins present on the tissue, so-called immuno-histochemistry (IHC), and hybridization of designed sequences of nucleotides to parts of chromosomes or genes (in-situ hybridization, ISH). Assessment can generally be done with a bright field microscope. Slides can be stored after investigation for a long period as back-up in case the diagnosis needs to be re-assessed.

With increasing understanding of the role of genetic mutations in cancer cells molecular diagnostics ("MDx") are becoming an essential part of pathology for selecting targeted therapies and predicting treatment response. This can be done by q-PCR micro-array and/or sequencing (Sanger or next generation) on the tissue. For the sensitivity and specificity of the MDx analyses it is of essential importance to select only relevant areas of the tissue slice. Dilution by non-cancerous tissue can lead to misdiagnosis.

Presently the pathologist marks with some kind of pen the area that needs to be selected for MDx. A lab technician then tries to remove the selected area from a new (thicker) slice of the same biopsy or even from the tissue block. This procedure is very inaccurate and cumbersome for two reasons: firstly the marking of the section is difficult to do by hand, especially if the image has been acquired by digital scanning. The pathologist needs to translate the image in his head to mark the slide by hand. Secondly, the technician needs to find a way to precisely scratch off the material without losing any or peeling off material outside the selected area. Moreover, contamination of the sample is a problem.

In summary, known procedures for sample extraction have one or more of the following drawbacks:

Cumbersome manual procedure.

Inaccurate selection of sample material from slide.

Risk of contamination due to manual procedure.

- Risk of misdiagnosis due to inaccurate selection.

Lack of documentation of selected material.

Lack of standardization and reproducibility.

In the following, an embodiment of the invention will be described that addresses the above issues and presents a new approach for an automated, flexible and precise selection of tissue from a standard microscopy slide. The first step in selective extraction of sample from a tissue section is the determination of the region of interest (ROI). This can be done based on histo-pathological staining procedures followed by inspection by a pathologist for diagnosis. Alternatively, the analysis can be supported by image analysis software tools or even completely be done automatically. Regions of interest can be characterized according to parameters derived from the image analysis. Depending on the kind of MDx analysis that is required according to clinical protocol the requirements for ROI definition can be adopted. After pathological selection of ROIs additional requirements can be taken into account to arrive at the final design of the ROI. The ROI file is then transferred to an irradiation device that projects the ROI (or the complementary surface) on the tissue slide that is covered by a photo-sensitive layer (called "photoresist" in the following). Upon illumination the solubility of the layer changes significantly.

The photoresist is thus patterned by selective irradiation. In a consecutive step the soluble area of the layer is dissolved by a solvent, thereby exposing the underlying tissue material that can be transferred by extraction and/or dissolution in the solvent for further purification, amplification and detection by PCR, sequencing or other molecular diagnostic technique. Preferably the projection of the ROI takes place without a mask and therefore does not require any preparation, cost or time. Any shape can be created at micro-meter resolution. The photoresist can be laminated on the tissue slide automatically inside the illumination equipment. Maskless projection technology is commercially available and used in practice in microlithography and image projection devices.

Figure 1 schematically illustrates an embodiment of the invention in a setup that is suited for the examination of a sample 11 of body tissue.

The examination starts at a sample preparation unit 100 in which a slice 11 of body tissue is prepared on a microscope slide 10 serving as a carrier. Typically, tissue samples are obtained by cutting a thin section of about 4-10 microns from a paraffin- embedded biopsy. The so-called coupes are placed on the microscope glass slide 10 on a water film to relax from the micro-toming strain and are then left to dry.

Moreover, the sample 11 may optionally be stained by an appropriate stain 12, e.g. by H&E or IHC. There are a number of staining protocols available for different applications. Staining protocols can be carried out on the bench manually by dipping the slide with the coupe in different solutions containing the reagents, but can also be performed in an automated fashion.

One or more markers M may be printed on or engraved in the microscope slide 10 that can later serve as reference points for relating image coordinates to actual coordinates on the slide 10. Moreover, the slide 10 with the sample 11 may optionally be covered with a cover slip (not shown) in order to allow for a high image quality during later scanning. After the preparation step, the slide 10 with the sample 11 is transferred to an image generating unit 200, which may particularly be a digital scanning microscope.

A digital image I of the sample is generated by the microscope 200 and communicated to a sample selection unit 300, which is here realized by a workstation 302 with a display (monitor) 301, a memory 303, and input devices 304 like a keyboard and a mouse. The image I of the sample can be displayed on the monitor 301 to allow for a visual inspection by a pathologist. The pathologist can identify a region of interest, R_ls in the image and mark it accordingly.

The identification of this "image-ROI" Ri may preferably be assisted (or completely be done) by automatic image analysis routines. The area can be optimized for total size etc., optionally taking constraints into account which arise from the sample extraction technology that is later applied (in the sample separation device 400). As a result of the calculation, an image-ROI, Ri, is determined that may be visualized on the screen 301 and/or in the microscope 200 while looking at the sample slide.

The image-ROI Ri can then be adjusted manually and selected by the pathologist with the aid of a cursor. Preferably, the pathologist can mark positions in any magnification. The marking will typically be done based on tissue morphology, which is the basis for deciding on the malignancy of the lesion or on the different cell types present. An unlimited number of images can be selected and marked. When finished, the software may provide an overview of the selected image-ROIs in the appropriate magnification for the pathologist and adjust the areas to a necessary resolution which can later be processed by a sample separation device 400 which takes care of the physical extraction of the sample for molecular testing. A file is created that contains the actual (image- and/or sample-) coordinates of the boundaries of the image-ROI Ri and the necessary references that can be interpreted by the sample separation device 400.

In the case the actual sample for MDx has been removed from a next slice of tissue and not from the one that was used for image analysis, a translation of the image-ROI has to be made by software using features of the next slice to overlay the image. Such features can be obtained from the next slice by an optical system optionally after a chemical staining reaction.

In the shown embodiment, the microscope slide 10 with the sample 11 is next transferred to the sample separation device 400 in which a sample-ROI, Rs, that corresponds to the selected image-ROI Ri is extracted from the remainder of the sample. Preferably, this sample-ROI Rs is transferred to a separate holder, for example a test tube 30. Moreover, the sample separation device may optionally be capable to extract several areas consecutively and submit those to separate molecular tests.

The sample separation device 400 will now be described in more detail. It comprises three essential units, namely:

- A coating unit 410 for coating the sample material 11 with a layer of a photoresist 20.

An irradiation unit 420 for selectively irradiating the photoresist 20.

An extraction unit 430 for removing the photoresist at the

sample-ROI only and extracting the sample material below.

The mentioned units may typically be realized in an integrated manner as a single device.

Figure 2 illustrates the above mentioned coating unit 410 schematically. The sample material 11 is provided as a layer on a microscope slide 10. With the help of e.g. a spray nozzle 411, the sample material 11 is coated with a thin layer of photoresist 20.

Alternatively, a photoresist layer can for example be applied via spin-coating of a solution containing the photosensitive material or by applying a solid (gel-like) layer of photosensitive material (e.g., Riston® MultiMaster Series Dry Film Photoresists of DuPont). Another option is the application of a so-called foil resist, that is resist which is in a rubbery state at room temperature and can be applied by lamination from a carrier substrate. In this way no contamination is created like with spin coating or spray coating.

Preferably, the coating is complete, i.e. covering the whole surface of the sample material.

Two types of photoresists can be used, positive and negative, transforming the material either into a soluble or insoluble material by irradiation. Such principles and materials are well known in the electronics industry. For a positive resist the sample-ROI will have to be illuminated, for a negative resist the area outside the sample-ROI. In the following, it will be assumed that a positive photoresist 20 is used. However, the

explanations apply mutatis mutandis to the usage of negative photoresists, too. After its application, the photoresist will typically cure to assume a solid consistency.

The application of the photoresist can be carried out inside an instrument that also provides the subsequent irradiation in a fully automated fashion. In the case of a dry resist the application will be done similar to an automatic cover slipping.

Figure 3 schematically illustrates the irradiation unit 420 in which the sample 11 on the microscope slide 10 that has completely been covered by the photoresist 20 is treated next. This treatment comprises a selective irradiation of the (positive) photoresist 20 within the sample-ROI Rs such that it becomes soluble. This soluble part of the photoresist at the sample-ROI Rs is denoted with reference sign "22", while the insoluble remainder has reference sign "21".

The projection of the image-ROI Ri onto the layer of photoresist 20 requires proper alignment. There are two main options of achieving that, depending on whether the identical slice of sample material is used that was used for the determination of the image-ROI Ri. In that case mechanical alignment can be used without additional imaging. In the case of a new slide with a neighboring slice of tissue the new slide needs to be imaged by the sample separation device 400 to recognize features that can be used for overlaying and where necessary interpolating the ROI taking into account small deformations and other scaling parameters for the best overlay possible. This can be a much less sophisticated image than the one used for the determination of the image-ROI, also depending on the required spatial resolution. In practice a resolution of several micrometers will be sufficient in the majority of cases.

Selective irradiation of the photoresist 20 might be achieved by disposing an appropriate mask between the photoresist and a light source. In the shown preferred embodiment, a maskless approach is used instead. A light beam L generated by a light source 421 is scanned by a mirror 422 and/or lenses over the surface of the photoresist 20. By controlling the light source 421 and/or the mirror/lenses 422 appropriately based on the available data of the image-ROI and its transformation into a sample-ROI, it can be achieved that only photoresist 21 above the sample-ROI Rs is irradiated. The mirror may for example be designed to scan the whole surface of the photoresist 20, and the light source 421 may be controlled such that it is switched off (or that its emission is blocked) whenever the resulting beam would reach a location that shall not be irradiated. Alternatively, a mirror and/or lenses may be controlled such that they direct light beams only to locations above the sample-ROI.

In practice, illumination in the irradiation unit 420 can be done with a UV light source 421, e.g. at a wavelength of about 365 nm. The image may be created for example based on a MEMS mirror array (using e.g. DLP® Technology of Texas Instruments). A latent image is thus created in the photoresist layer that is provided on the sample slide.

After illumination the soluble part 22 of the photoresist 20 can be removed by immersion in a solvent serving as developer solution that also extracts the nucleic acids (NA) and optionally lyses the biological sample in the accessible part. Certain developing solutions (solvents) do not interfere with the next step of NA purification, which is done in standard equipment available in clinical laboratories as the first step of MDx analysis. Both, acidic as well as basic developers are available and induce cell lysis. Optional heating steps can be applied before and after illumination and during development of the resist as to achieving the optimal contrast in solubility and extraction performance.

Figure 4 schematically illustrates an embodiment of the extraction unit 430 that can be used for the aforementioned steps. The slide 10 is immersed in a solvent 431 for removing the soluble photoresist and extracting the accessible part of the sample. To limit the volume of solvent and for reasons of convenience a preferred embodiment uses a cover 25 that is placed above the photoresist 20 and has openings to introduce/remove the solvent.

The slide 10 with the cover 25 may preferably be placed on a hotplate 433 and brought to the desired temperature for complete lysis and/or extraction of nucleic acids and optionally melting of the paraffin in the case the sample is a formalin- fixed paraffin embedded (FFPE) tissue or cytology sample. Upon heating the chemical substances of interest will dissolve in the exposed part of the sample and - in the case of an FFPE sample - the paraffin will be phase-separated from the solvent. FFPE samples that require imaging to determine the sample-ROI do not contain the paraffin anymore, as they have be

deparaffmated before staining already. Before, or during or after cooling down the solvent containing the sample can be pipetted off or poured into an Eppendorf test tube 30 or transferred otherwise to a molecular examination unit 500 for purification, amplification, and detection of the desired biomarkers. In a preferred embodiment the solvent containing the sample is pumped into a collection chamber by microfluidic actuation. Such actuation can be created by a pneumatic interface with the cover or other microfluidic actuation principle. Passive and/or active actuators can be integrated in the cover to from an integrated microfluidic flow system that can also be used to introduce the solvent from a supply chamber into the sample chamber and/or to actuate the solvent during the

developing/extraction phase to enhance the solution process.

In a preferred embodiment (focused) ultrasound may be applied from an ultrasonic transducer 432 to accelerate cell lysis/extraction.

A new image may be scanned from the tissue slide 10 before and/or after extraction of the sample-ROI Rs to confirm and control the selection. This image may be archived on the workstation 302 together with the original image and the results of the MDx analysis.

The microscope slide 10 with the remainder of the sample can optionally be stored in a storage unit 600 for later access and verification, or it may simply be discarded. Later processing steps with the slide 10 may particularly comprise a follow up examination comprising for example at least one new (particularly different) staining and/or at least one new (particularly different) molecular assay with another region of interest.

In the molecular examination unit 500, several molecular techniques may be available for analysis of the selected sample ROI, like PCR (several techniques are comprised under this term, like q-PCR, RT-PCR, qrt-PCR, digital PCR, etc.) for detecting single point genetic mutations in cancer cells or any other DNA-mutation, DNA-deletion, DNA- insertion, DNA-rearrangements or copy number amplification or other structural change, and/or determining the degree of RNA expression of genes or other transcribed DNA sequences in cancer cells, or RNA or DNA sequencing (next gen sequencing) for determining a wider spectrum of genetic variations in the cancer cells, for example either on the whole genome, or the whole exome, or targeted to smaller regions of the genome, to the exosome, or the transcriptome, and in various depths. The result is interpreted in terms of genetic mutations of the cancer cells and their corresponding RNA expression profiles which are relevant for the prognosis or alternatively the susceptibility to certain treatment, like by targeted drugs, or to actually assess the effect of a treatment already started or finished (treatment monitoring).

Experimental results

In an experiment sections of FFPE treated SKBR3 cells were processed according to the above described method. Photoresist was spray-coated onto these cells. After illumination with a 365 nm UV lamp through a contact mask with a square aperture the slide was exposed to a buffer solution according to a standard protocol for mRNA extraction from tissue (Qiagen). Sample extracted according to the proposed method was compared to sample that was removed without the use of a photoresist (positive control) and sample that was treated with a standard developer solution suggested for this photoresist.

Figure 5 shows a diagram with the results that are expressed in threshold values, C_T, of the qRT-PCR. The expression of Her2 mRNAs was investigated and compared to that of GAPDH which is a house-keeping gene. The proposed method (sample #5), indicated as "0.025N PKD protK", yields comparable C_T values as sample #1 which was introduced directly without lithographic selection, while sample #4, indicated as "0.025N protK", which was obtained with a standard developer, did not yield a PCR result. In sample #2 ("0.25N") and #3 ("0.025N"), development of the photoresist and extraction of the sample material were done in separate steps with different media. This shows that the method works as proposed and allows a simplified, automatic solution. The detailed protocol of the experiments was as follows:

After the sample slide is inserted in the sample separation device 400, the following steps were executed:

- photoresist layer (20) (e.g. "MicroSpray™ Positive" of MicroChem,

Germany) with cover (25) is provided on sample slide;

- a sample-ROI is determined (this was an arbitrary region for the purpose of this experiment; during clinical usage, a reference on the slide may be imaged by a camera and the sample-ROI position may be calculated);

- sample-ROI is illuminated through a mask (or with maskless technology);

- extraction medium (depending on kind of experiment) is pipetted and oscillated/actuated;

- the slide is heated for required time;

- slide with cover is taken from the bench.

Next, the slide is processed by a lab technician:

- buffer containing the sample is pipetted off from cartridge;

- sample in buffer is injected in barcoded eppendorf tube;

- tube is transferred to standard sample prep station in MDx lab.

Five variants #l-#5 of the above general procedure were carried out on FFPE material of SKBR3 (4 μιη thick) according to the following protocols:

#1 #2 #3 #4 #5

3^X xylene 8 min 3^X xylene 8 min 3^X xylene 8 min 3^X xylene 8 min 3^X xylene 8 min

100% EtOH 100% EtOH 100% EtOH 100% EtOH 100% EtOH 5 min & 30 sec 5 min & 30 sec 5 min & 30 sec 5 min & 30 sec 5 min & 30 sec

Photoresist Photoresist Photoresist Photoresist sprayed sprayed sprayed sprayed

1 min UV 1 min UV 1 min UV 1 min UV (365nm) (365nm) (365nm) (365nm) - 15" NaOH Γ NaOH - - 0.25N 0.025N

- Wash in MQ Wash in MQ - -

Apply cover Apply cover Apply cover Apply cover Apply cover

(hybridization (hybridization (hybridization (hybridization (hybridization chamber) chamber) chamber) chamber) chamber)

Add 16 μΐ Add 16μ1 protK Add 16μ1 protK Add 10μ1 0.25Ν Add 10μ1 0.25Ν protK + 84μ1 + 84μ1 PKD + 84μ1 PKD NaOH + 16μ1 NaOH + 16μ1 PKD protK + 74 μΐ protK + 74 μΐ

MQ PKD

15 min 60°C 15 min 60°C 15 min 60°C 15 min 60°C 15 min 60°C

(EtOH: ethanol; MQ: milli-Q (water); protK: proteinase K (QIAGEN); PKD: buffer (proprietary QIAGEN))

With each sample, molecular diagnostics was done according to standard protocols. The results of this are shown in Figure 5.

In summary, a protocol has been described that allows the extraction of material from a tissue or cytology slide into a solution in a single step making use of a photolithographic mask. The region of interest (ROI) is selected based on image analysis and interpretation of stained tissue. The stained tissue is covered by a thin photo-sensitive layer e.g. by laminating or spraying. The ROI file is transferred to an illumination device that projects the ROI (or the complementary surface) on the photo-sensitive layer. Upon illumination the solubility of the layer changes significantly. In a consecutive step the illuminated area of the layer is treated with a solution that dissolves the photoresist in the ROI only and simultaneously extracts the genetic material (RNA, DNA) from the underlying sample in the ROI in a single step. The components of the resist that are dissolved do not interfere with the downstream processing of the sample for MDx analysis. MDx analysis may be done to determine genetic characteristics, like mutations and/or mRNA expression profiles by PCR, sequencing or other molecular diagnostic technique. The approach can be applied in molecular pathology, in particular for patient stratification in oncology based on

identification of molecular changes in cancer cells. An essential step is that the development of the resist and the extraction of the sample material takes place in a single step by a single reaction solution.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless

telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope.

Claims

CLAIMS:

1. A method for the separation of material from a region of interest (Rs), in a biological sample (11), said method comprising the following steps:

a) coating the sample (11) at least partially with a layer of a photoresist (20);

b) irradiating said layer selectively such that the photoresist (20) is soluble at the sample-ROI (Rs) and insoluble elsewhere;

c) applying a solvent (431) to the photoresist (20) that dissolves the soluble photoresist (22) and that extracts sample material below the dissolved photoresist, characterized in that the solvent (431) comprises at least one of

a base, particularly NaOH or KOH;

a buffer;

a detergent;

a biological agent, particularly a proteinase;

a chaotropic salt.

2. A sample separation device (400) for the separation of material from a region of interest (Rs), in a biological sample (11), comprising:

a coating unit (410) for coating the sample (11) at least partially with a layer of a photoresist (20);

an irradiation unit (420) for irradiating said layer selectively such that the photoresist (20) is soluble at the sample-ROI (Rs) and insoluble elsewhere;

- an extraction unit (430) for applying a solvent (431) such that the soluble photoresist (20) is dissolved and the sample material below is extracted,

characterized in that the solvent (431) comprises at least one of a base, particularly NaOH or KOH;

a buffer;

- a detergent;

a biological agent, particularly a proteinase;

a chaotropic salt.

3. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that the base has a concentration of about 0.002 mol/1 to about 0.30 mol/1.

4. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that the photoresist (20) is a negative photoresist.

5. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that a light beam (L) is directed only to locations of the photoresist (20) at the region of interest (Rs), or that it is alternatively directed only to locations outside the sample-ROI.

6. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that a molecular assay (MDx) is executed with sample material obtained from the region of interest (Rs), particularly an assay comprising PCR and/or sequencing.

7. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that the sample (11) is disposed on a carrier, particularly on a microscope slide (10).

8. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that a cover (25) is disposed above the region of interest (R_s).

9. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that an actuator (432) is provided for applying energy such as electromagnetic radiation, heat and/or ultrasound to the sample.

10. The method according to claim 1 or the sample separation device (400) according to claim 2,

characterized in that the region of interest (Rs) is derived from a second region of interest (Ri), in an image (I) related to the sample, particularly an image of the sample itself or of an additional sample generated from the same material as the sample.

11. The method according to claim 1 or the sample separation device (400) according to claim 2

characterized in that an image of the sample (1 1) is generated before and/or after removal of sample material from the region of interest (Rs).

12. The method or the sample separation device (400) according to claim 6 and 12,

characterized in that the results of the molecular assay and properties of the second region of interest (Ri)are reported and interpreted in a combined fashion for medical diagnostics.

13. A solvent (431) for usage in a method according to claim 1 or a sample separation device (400) according to claim 2, comprising at least one of:

a base, particularly NaOH or KOH;

a buffer;

a detergent;

a chaotropic salt;

- and a biological agent, particularly a proteinase.