METHOD

The invention relates to a method of quantitative immunohistochemical determination of tissue antigens.

Prior art

The immunohistochemical technique on histological sections enables cell antigens to be localised in tissues, giving rise to a coloured reaction visible under the microscope. Said procedure is currently used for diagnostic purposes, finalized both to morphological and functional tissue characterization, and to determine the therapeutic approach. The presence and amount of markers, such as receptors in particular, is the prerequisite that predicts the response to targeted treatments. Hence the need to “quantify” immunohistochemical staining. Said quantitation is currently expressed with a visual evaluation of the relative frequency of specifically stained cells (1+, 2+, 3+), with highly detailed counts (e.g. no. of positive cells in 1000 cells) and, most recently, with computerised image analysis techniques. Said procedures are not only complex, but also poorly replicable as they are liable to subjective interpretations. Immunohistochemical methods, which were developed to enable antigens to be localised in cells and tissues, are based on the antigen-antibody bond, leading to a stained reaction giving a microscopic display of the binding site. The display of the reaction, initially based on fluorescent stains (hence the term “immunofluorescence”), was subsequently developed by using antibodies conjugated with enzymes, in particular horseradish peroxidase, and the reaction site is then displayed with an enzyme-histochemical reaction giving rise to an insoluble coloured product. The main reagent used for this purpose is diaminobenzidine, which acts as acceptor of the oxygen released following the reaction of the peroxidase enzyme on hydrogen peroxide. Diaminobenzidine is currently used in immunohistochemistry (IHC) because, following oxidation, it gives rise to a brownish- black insoluble product that precipitates on the site of production and labels the cells containing the antigen. The techniques have evolved with time, and other enzymes (alkaline phosphatase, glucose oxidase and beta-galactosidase) have been used as an alternative to peroxidase; in all such cases other substrates were used, all of which have the common property of giving rise to insoluble coloured products, that microscopically label the various cells containing the antigen tested for.

This type of approach, based on identification of particular cell differentiations and localisation of antigens labelling differentiating lines, led to considerable progress in histological and cytological interpretation and had considerable diagnostic repercussions, especially in the oncological field, allowing the various types of tumour to be characterised not only on a morphological and structural basis, but also on a functional and differentiating basis. A further, significant development took place when it was observed that immunohistochemical reactions could also be conducted on tissue sections fixed in formalin and embedded in paraffin, because physicochemical treatments, in particular heating of sections, gave rise to “antigen retrieval”. It was also observed that not only the presence of a given antigen, but also its amount of expression, allowed the appropriate pharmacological treatment to be devised. In particular, in breast cancer, it was observed that the amount of expression of the HER2 receptor was the factor that allowed the response of a tumour to targeted anti-HER2 treatments to be predicted (Taylor, 1993; Grube, 2004; Taylor and Levenson, 2006; Taylor, 2014). In currently approved protocols, treatment with anti-HER.2 antibodies is reserved for cases wherein HER2 expression is highest (known as 3+), whereas the treatment is ineffective if expression is lower (value 0, 1, 2+). The presence, and above all the amount, of expression of different antigens (“markers”) represents a crucial factor that influences the use of specific procedures in the ambit of tailored treatments (Lehr et al., 2001; Hatanaka et al., 2001; Rhodes et al., 2002). At present, conventional H4C is therefore a powerful tool for pathological diagnosis; however, it has intrinsic limitations in the case of tailored treatments, which increasingly often require reliance on the quantitative measurement of biomarkers. Customised treatments require a solid stratification of patients into prognostic and predictive categories based on reliable information of various kinds: clinical information and radiological, pathological and molecular tests. It is often necessary to combine several test procedures to obtain the optimum results. Protein biomarkers, measured in pathological tissues, are important because they represent molecules that constitute the final result of genetic anomalies and metabolic disorders, and can become the targets of treatment. The need to determine the amount of localised antigen using immunohistochemistry as a tool for quantifying protein biomarkers in tissue is currently pursued, because clinical oncologists require this information to devise the treatment approach (Becker, 1993; Cross, 2001; Seidal et al., 2001). Determining the amount of stain deposited on the sections by microscopic examination is subjective and poorly replicable, which conflicts with the need for highly reliable data in order to make decisions on the adoption of specific treatments. Numerous attempts have been made using manual counts of positive cells compared with a fixed number (100 or 1000) of the total cells, or using image analysis on electronically acquired (“scanned”) preparations, but even such approach has given somewhat unreliable results, due to both technical difficulties and difficulty of interpretation (Cross, 2001; Di Cataldo et al., 2012; Linden et al., 2015; Laurinavicius et al., 2016). The various methods, options and limitations of said procedures were illustrated by Taylor (2014). The amount of the problems and opportunities arising in quantitative H4C is very broad, because reaction times and temperature, as well as the thickness of the sections, can affect the intensity of the staining. Moreover, a true reference standard for calibrating and checking the analytical accuracy of the test is difficult to obtain with IHC. A possible method could involve using a quantitative internal reference standard based on double staining in IHC to compare the amount of an intrinsic tissue. Moreover, although the bright-field chromogen produced in IHC (generally DAB) clearly delineates the tissue architecture and is preferred by pathologists, immunofluorescence-based techniques are more suitable for detection of the multiplex antigen than for true quantitation. As IHC does not provide data on the real protein concentrations, only information about the extent of the positive areas and cells, it derives that the data supplied are highly subjective and poorly replicable. Finally, it should be added that production of the stained product (oxidised DAB) deposited on the reaction site does not increase linearly with the reaction time, but reaches a plateau, after which the accumulated stain prevents further development of the reaction by steric impact. Finally, it should be specified that integral cells or whole nuclei are not evaluated in histological sections, but sections thereof, which may be equatorial, but are more often only tangential (the cells have a variable diameter, ranging between about 7 and 15 microns, while the sections have a thickness of about 4 microns). Hence the difficulty, and perhaps impossibility, of measuring the percentages of cells or nuclei positive for a given reaction, compared with the rest of the negative cells or nuclei.

Description of the invention

An innovative process of antigen quantitation has now been found, characterised by the development of histological sections processed by immunohistochemistry by means of a reaction with a coloured product quantifiable on the basis of spectrophotometric absorption. The value obtained from the spectrophotometer reading is calculated as a ratio of a value obtained by a second immunohistochemical procedure on the same section, by labelling a general indicator of the amount of cells or reference structures (such as epithelial cells or total nuclei present in the same section).

The invention therefore provides a method of quantitative immunohistochemical determination of tissue antigens that comprises: a) Placing the histological preparation in contact with an anti-antigen tissue antibody of diagnostic interest, and then with an anti-antibody antibody labelled with a specific enzyme for a substrate detectable by colorimetry; b) Developing the preparation with a substrate that gives rise to a soluble product determinable by spectrophotometry, and reading of a first signal X; c) Removing the anti-antigen and anti-antibody antibodies after step b) by washing to remove the specific binding; d) Placing the washed preparation in contact with a second anti-epithelial cell or anti-nuclear marker antibody, and then with an anti-antibody antibody labelled with a specific enzyme for a substrate detectable by colorimetry; e) Developing the preparation with a substrate that gives rise to a product determinable by spectrophotometry, and reading of a second signal Y; f) Determining index X/Y x 100.

The method according to the invention therefore provides two values, relating to the same section: the amount of reference marker, and the amount of the reference cells (total cells. The marker/cell ratio will indicate, in numerical values, the percentage of cells positive for the marker and the intensity of expression thereof. The result will therefore be an objective numerical value that measures the expression of a given antigen of interest, as a ratio of the total number of tumour cells. Similarly, the percentage of cells or tissue components (nuclei, connective tissue, blood vessels or inflammatory infiltrate) can be measured in relation to a reference component (amount of DNA or cytoplasm or connective tissue present, using suitable antibodies).

The specific enzyme for a substrate detectable by colorimetry is preferably a peroxidase, such as horseradish peroxidase.

Examples of soluble substrates for peroxidase include tetramethylbenzidine (3, 3', 5, 5' tetramethylbenzidine, TMB), ABTS (2,2'-azino-di[3-ethylbenzothiazoline] sulphonate) and OPD (o-phenylenediamine) which give a blueish or greenish soluble product that accumulates in the reaction medium according to the reaction time and the amount of peroxidase present. Said reaction enables the amount of antigen present to be evaluated indirectly by spectrophotometric measurement of the amount of colour produced.

As an alternative to peroxidase, other enzymes, such as alkaline phosphatase, glucose-oxidase and beta-galactosidase, can be used, subsequently employing soluble coloured reagents evaluatable with spectrophotometry or fluorescent products, also detectable with a spectrophotometer. The development can also be performed with reagents that give rise to coloured products which are water-insoluble but subsequently solubilisable in solvents, and can therefore always be red with the spectrophotometer.

The method according to the invention is suitable for quantitation of any tissue antigen of interest for diagnostic, prognostic and therapeutic purposes. A particularly interesting field of application relates to tumour antigens such as HER2, Ki67 and PD-L1. Examples of cell markers usable according to the invention are cytokeratin, EMA (Epithelial Membrane Antigen) and dsDNA.

By following similar procedures, it will be possible to use the quantitative analysis described herein to evaluate different cell types present in the same section comparatively (e.g. the ratio between T lymphocytes and B lymphocytes), each cell type being identified by specific markers.

The method according to the invention can be performed not only on the same section but also on parallel sections, such as parallel serial sections.

The results can be read not only with conventional spectrophotometry techniques, but also by scanning and total byte weight of the scanned image.

If desired, the total amount of proteins present in a histological section can be evaluated by lysing it with Lysis Buffer or SDS (sodium dodecyl sulphate) elution, and measuring the proteins in the lysate by Lowry reaction or biuret reaction.

The total DNA present in a histological section can be evaluated by lysing the section (e.g. with peptidase K) and then reading the amount of DNA on the basis of the spectrophotometric absorption.

Detailed description of the invention

For example, to establish the percentage of neoplastic cells positive for antigen X (e.g. HER2) in a breast cancer, and also the intensity of expression of said antigen, the immunohistochemical reaction for the antigen can be performed with an anti-antigen X primary antibody, followed by a peroxidase-labelled antibody, against the primary antibody. At this point, instead of developing with DAB (as routinely performed), the histological section is exposed to a fixed amount (e.g. 0.5 ml) of TMB-based reagent. After a set time (e.g. 10 min.), the development reagent is taken up and the intensity of reaction evaluated spectrophotometrically at the established wavelength to obtain a numerical value (“value X”). The resulting numerical value is not significant per se. However, it acquires a useful value if related to the basal value determinable by conducting steps c)-f) of the method according to the invention. In particular, the histological section used in steps a) and b) is washed in saline solution, then treated with acid solution at pH 2.2 for 10 min, to entirely remove the antibodies bonded in the first reaction. The section undergoes a new immunohistochemical reaction, using antibodies against antigen Y (e.g. anti-keratin, which stains all the epithelial cells of the carcinoma listed above). This will be followed by peroxidase-labelled antibodies against the primary antibody. By developing the reaction with TMB as described above, and evaluating the intensity of the reaction with the spectrophotometer, a numerical value (“value Y”) is obtained. By calculating the ratio between value X and value Y (X/Y x 100) a reference value (“Index”) is obtained, which indicates the corresponding number of tumour cells expressing antigen X and the intensity of said expression, in relation to the total number of tumour cells. Said value will be independent of the width or thickness of the section. It will thus be possible to provide to the clinicians numerical values of all the targets that indicate possibilities of a therapeutic response.

In order to expose the histological sections (on a slide) to the correct amount of TMB medium for incubation, the use of glass or plasticwells, slightly longer than a histological slide, is preferred, e.g. width 25 to 27 mm, length 75 to 78 mm and height 1 to 5 mm, preferably 25 x 76 mm, with a depth of about 3 mm. Said wellsenable the slides to be incubated face down, thereby directly exposing the histological sections to the incubation medium. At the end of incubation, the slide is removed and the medium remains in thewell, from which it can be removed for measurement. The wells wherein the preparations are incubated can also be vertical, with a width of 1 to 3 mm and height of 50 to 75 mm. Incubation can also be performed by placing the incubation medium in contact with the histological sections, over or under the slide or between the histological slide and a coverslip. The following examples illustrate the invention in greater detail.

Example 1

6 cases of breast cancer with the following characteristics were selected:

On immunohistochemical staining for HER2, on the basis of the extent and intensity of the reaction for HER2 on the tumour cell membrane, cases 19584 and 20926 were scored as 3+, case 20574 as 2+ and case 18980 as 1+, while cases 18583 and 24520 were wholly negative (o+). 4 micron thick histological sections were taken from each case, and mounted on histological slides.

The sections were processed immunohistochemically, after paraffin removal, using a Ventana automated apparatus (Ventana BenchMark AutoStainer, Ventana Medical Systems, USA). After antigen retrieval treatment with Cell Conditioning 1 (CC1, slightly basic; Ventana) for 36 minutes at 95°C, the sections were incubated with 4B5 rabbit antibody (Roche). After washing, the sections were treated with peroxidase- labelled anti-rabbit immunoglobulin antibody (Ultraview detection kit, Ventana).

The sections did not undergo the usual routine DAB staining, but were placed in phosphate-buffered saline solution (PBS) and processed with reagent containing 3, 3’, 5, 5’- tetramethylbenzidine (TMB), liquid substrate for ELISA (manufactured by Sigma, Milan). We used glass or plastic wells with dimensions suitable to contain a histological slide (2.5 x 7.5 cm) and a depth of 3 mm. We placed 0.5 ml of TMB reagent in saidwell , then immersed the histological section face down, ensuring that the section was fully in contact with the reagent. The reaction was developed (in the dark) for 10 min. at room temperature, and the section was then replaced in PBS. The reaction medium was collected, mixed in equal parts with blocking liquid (pH 2), and processed for microspectrophotometer reading. The histological sections, after washing in PBS, were immersed in acid solution (0.01N HC1, pH 2.2) for 10 min. at room temperature, then washed again in PBS and processed immunohistochemically in a Ventana apparatus as specified above. After treatment with protease 1 for 4 min, the sections were treated with anti-PanCytokeratin mouse antibody (AE1/AE3/PCK26, Roche), then with a reagent designed to detect mouse immunoglobulins (Ultraview detection kit, Ventana). The sections, collected in PBS, were treated with TMB as described above, to measure the amount of peroxidase and therefore, indirectly, the amount of cytokeratin present in the sections, as an indicator of the epithelial cells present. After the reaction, the TMB reagent was processed for reading on the spectrophotometer. After development of the reaction in TMB, the sections were washed in PBS, then stained for peroxidase with diaminobenzidine (DAB) according to the ordinary immunohistochemical staining process. This enabled the correctness of the reaction to be checked visually, because uniform cytoplasmic staining of all breast cancer epithelial cells is observed. The absence of tumour cell membrane staining confirmed the removal (by acid solution) of the anti- HER2 antibodies bonded during the first step of the process.

When the TMB reagent was read on the spectrophotometer, the following HER2 values were obtained:

Pan Cytokeratin readings

The individual values are obviously not significant per se. However, when the percentage ratio HER2/cytokeratin X 100 is calculated for the individual cases, the following values are obtained, namely a HER2/ CK index: 0,25/2,52 x 100= 9,90%

0,52/3,20 x 100= 16,00%

1 ,32/3,10 x 100= 42,50%

0,12/2,83 x 100= 4,20%

2,25/2,35 x 100= 95,70% 1 ,43/2,29 x 100= 62,70%

It was accordingly found that the two cases which, on immunohistochemical reading, had been evaluated as 3+ (cases 19584 and 20926), had the highest values (62 and 95%), case 20574 (2+) had an intermediate value (46%) and case 18980 (1+) a value of 16%, while cases 18583 and 24520 (o+) had the smallest percentage values (9 and 4%).

Example 2

An immunohistochemical method has been designed to detect the DNA present in a histological section using an anti-double-stranded DNA mouse monoclonal antibody (Double Stranded DNA Monoclonal Antibody (AE-2) Catalogue number: MAI 35346; manufacturer Thermo Fisher Scientific, Monza, Italy). Using a suitable dilution of the antibody in buffered saline solution (PBS) and employing immunohistochemical methods with peroxidase-labelled anti-mouse IG antibodies (Ultraview detection kit, Ventana), and developing the immunohistochemical reaction with diaminobenzidine (DAB), staining of all the nuclei present in the section was observed. Two cases of breast cancer were selected. After fixing in buffered formalin and embedding in paraffin, the sections were stained with antibodies against Ki67, a nuclear marker expressed in proliferating cells. In said immunohistochemical reaction, the number of tumour cell nuclei positive for Ki67 is taken as reference number to indicate the proliferative activity in the tumour. In these two cases (case A and case B), the number of positive Ki67 cells was calculated (optical evaluation) as 10% in case A, and 80% in case B. Sections of the two cases were processed immunohistochemically, after paraffin removal, using a Ventana automated apparatus (Ventana BenchMark AutoStainer, Ventana Medical Systems, USA). After antigen retrieval treatment with Cell Conditioning 1 (CC1, slightly basic; Ventana) for 36 minutes at 95°C, the sections were incubated with anti-Ki67 MIB1 mouse antibody (Roche). After washing, the sections were treated with peroxidase-labelled anti-mouse immunoglobulin antibody (Ultraview detection kit, Ventana). The sections were placed in phosphate-buffered saline solution (PBS) and processed with reagent containing 3, 3’, 5, 5’- tetramethylbenzidine (TMB), liquid substrate for ELISA (Sigma, Milan), by the method reported above. On the spectrophotometer reading, values of 0.88 were obtained in case A and 1.63 in case B. The histological sections, after washing in PBS, were immersed in acid solution (0.01N HC1, pH 2.2) for 10 min. at room temperature, then washed again in PBS and processed immunohistochemically in a Ventana apparatus as specified above. The sections were treated with double-stranded DNA monoclonal mouse antibody (Double Stranded DNA Monoclonal Antibody (AE-2)), then with reagent designed to detect mouse immunoglobulins (Ultraview detection kit, Ventana). The sections, collected in PBS, were treated as above with TMB, to measure the amount of peroxidase and therefore, indirectly, the amount of dsDNA (i.e. the total nuclei present in the sections, as an indicator of the cells present). After the reaction, the TMB reagent was processed for reading on the spectrophotometer. We obtained the following values: Case A = 3.28; Case B= 3.15. These values indicate that although the sections had almost identical dimensions, the number of nuclei present was different. By calculating Ki67 value / DNA value x 100, the following results were obtained: case A: 0.88/ 3.28 x 100 = 26.8; case B: 1.63/ 3.15 x 100 = 51.7. The data indicate the possibility of obtaining a numerical evaluation (index) of the expression of antigen Ki67, which clearly differs in low-proliferation and high-proliferation tumours.

Example 3

Tissue cancers are currently tested for expression of PD-L1 (Programmed Death- Ligand 1) in order to devise the therapeutic approach. It has been observed that in immune “checkpoints” PD1-PDL1 there is a mechanism that blocks the immune response, and that expression of PD-L1 is correlated with immune evasion by tumour tissues. Treatment with anti-PD-Ll antibodies can have the effect of controlling tumour growth in tumours that express said marker, and the therapeutic effect observed is correlated with the intensity of expression. PD-L1 is therefore tested for in histological sections, using immunohistochemical methods. The evaluation of intensity of expression is reported as the percentage of positive cells.

Currently, testing for PD-L1 expression is performed in numerous types of tumours, especially lung cancers and malignant melanomas (Scheel et al., 2016; Sunshine et al., 2017). If the percentage measurement method relating to marker PD-L1 is to be used, it is essential to identify the reference marker, expressed in all tumour cells. Said marker can be identified with cytokeratin in the case of carcinoma, whereas in the case of melanoma, protein SI 00 is preferred.

Two cases of malignant skin melanoma were selected. Sections of said cases (fragments of tumour tissue, after fixing in buffered formalin and embedding in paraffin) were stained with anti-PD-Ll antibody (SP263). In said immunohistochemical reaction the number of tumour cells positive for PD-L1 on the cell surface is taken as the reference number. The number of positive PD-L1 cells (optical evaluation) was 3% for case A and 30% for case B. Sections of the two cases were processed immunohistochemically, after paraffin removal, using a Ventana automated apparatus (Ventana BenchMark AutoStainer, Ventana Medical Systems, USA). After antigen retrieval treatment with Cell Conditioning 1 (CC1, slightly basic; Ventana) for 36 minutes at 95°C, the sections were incubated with anti-PD-Ll (SP263) antibody (Roche). After washing, the sections were treated with peroxidase-labelled anti-immunoglobulin antibody (Ultraview detection kit, Ventana). The sections were placed in phosphate-buffered saline solution (PBS) and processed with reagent containing 3, 3’, 5, 5’- tetramethylbenzidine (TMB), liquid substrate for ELISA (Sigma, Milan), by the method reported above. On the spectrophotometer reading, values of 0.55 were obtained in case A and 1.54 in case B. The histological sections, after washing in PBS, were immersed in acid solution (0.01N HC1, pH 2.2) for 10 min. at room temperature, then washed again in PBS and processed immunohistochemically in a Ventana apparatus as specified above. The sections were treated with anti-S-100 protein antibody, then with a reagent designed to detect immunoglobulins (Ultraview detection kit, Ventana). The sections, collected in PBS, were treated as above with TMB, to measure the amount of peroxidase and therefore, indirectly, the amount of S-100 protein as an indicator of the cells present. After the reaction, the TMB reagent was processed for reading on the spectrophotometer. The following mean values were obtained: Case A = 3.25; Case B= 3.10. These values indicate that although the sections had almost identical dimensions, the number of cellspresent was different. By calculating PD-L1 value / S100 value x 100, the following results were obtained: Case A: 0.55/ 3.25 x 100 = 16.9. Case B: 1.54/ 3.10 x 100 = 49.6.

The data indicate the possibility obtaining a numerical valuation (index) of the expression of antigen Pd-Ll, which clearly differs in low-expressing and high-expressing tumours, which are therefore more likely to respond to targeted treatment.

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