Stratification of patients for assessing the suitability of a therapy

Abstract

A method for stratification of a patient for assessing suitability of a therapy for the patient suffering from an ER (estrogen receptor) and/or PR (progesterone receptor) positive and HER2 (human epidermal growth factor receptor 2) negative cancer, the therapy being directed towards a signaling pathway, includes: (i) determining an activation status of an ER and/or PR signaling pathway by applying a Proximity Ligation Assay to detect in a tissue sample of the patient a presence of at least one member of the ER family, the at least one member being part of a transcription factor complex, and at least one protein selected from a group consisting of TAFs (TATA-binding protein associated factor), TBP (TATA-box binding protein), POLII (RNA polymerase II), TFII (transcription factor H), p300, CREB (cyclic-AMP response element-binding protein), and CBP (CREB binding protein), wherein the at least one protein is part of the same transcription factor complex.

Claims

1. A method for stratification of a patient for assessing suitability of a therapy for the patient suffering from an ER (estrogen receptor) and/or PR (progesterone receptor) positive and HER2 (human epidermal growth factor receptor 2) negative cancer, the therapy being directed towards a signaling pathway, comprising: (i) determining an activation status of an ER and/or PR signaling pathway by applying a Proximity Ligation Assay to detect in a tissue sample of the patient a presence of at least one member of the ER family, said at least one member being part of a transcription factor complex, and at least one protein selected from a group consisting of TAFs (TATA-binding protein associated factor), TBP (TATA-box binding protein), POLII (RNA polymerase II), TFII (transcription factor II), p300, CREB (cyclic-AMP response element-binding protein), and CBP (CREB binding protein), wherein the at least one protein is part of the same transcription factor complex, wherein the stratification is based on the activation status determined in (i) and the suitability of the therapy is assessed based on the stratification, wherein a signaling pathway of (i) is determined to be active, if the at least one ER family member is located in the nucleus in the Proximity Ligation Assay close proximity to the at least one protein selected from the group consisting of either TAFs, TBP, POLII, TFII, p300, CREB, and CBP.

2. The method of claim 1, wherein the at least one member of the ER family is an ER dimer.

3. The method of claim 1, additionally comprising: (ii) determining the activation status of a signaling pathway, which is different from the signaling pathway assessed in (i), by applying an in-situ staining assay to detect in the same or in a different tissue sample of the patient the presence of at least one member of the ER family and at least one protein selected from the group consisting of, c-Fos, c-Jun (AP1), SP1, CREB, GATA-transcription factor, NFkappaB family, c/EBP (CCAAT/enhancer binding protein) proteins, FOXO (Forkhead Box 0), SMAD transcription factor proteins, wherein the stratification is further based on the activation status determined in (ii).

4. The method of claim 1, additionally comprising: (iii) determining the phosphorylation status of at least one member of the ER family at serine 305 in the tissue sample of the patient, wherein the stratification is further based on the phosphorylation status determined in (iii).

5. The method of claim 1, additionally comprising: (iv) determining the activation status of one or more signaling pathways, which is/are different from the signaling pathway assessed in (i) and is/are selected from the group consisting of P13K (Phosphoinositide 3-kinase) pathway, Wnt pathway, hedgehog (HH) pathway, NFkappaB, Notch pathway, TGFbeta, FGF, VEGF, EMT, pathways, other nuclear receptor pathways, like the AR, RAR, PPAR, glucocorticoid, VitD pathways and growth factor pathways, like insulin GF and EGF, by applying an in-situ staining assay, wherein the same or a different tissue sample of the patient is used, wherein the stratification is further based on the activation status determined in (iv).

6. The method of claim 1, additionally comprising: (v) determining the activation status of the P13K pathway by applying an in-situ-staining assay to detect in the same or in a different tissue sample of the patient an active PKB (protein kinase B)/Akt (protein kinase B) protein by measuring the phosphorylation status of PBK/Akt, wherein the stratification is further based on the activation status determined in (v).

7. The method of claim 1, additionally comprising: (vi) applying an in-situ staining assay to detect in the same or in a different tissue sample of the patient the presence of at least one member of the ER family and at least one component which is expected to be present at any ERE (Estrogen Response Element) half-site or any non ERE site and/or wherein the at least one component is selected from the group consisting of AP1, cAMP response element-binding protein/CREB, GATA transcription factor, NFkappaB, SP1, C/EBP, FOXO, and SMAD transcription factor proteins, wherein the stratification is further based on the detection in (vi).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the following drawings:

(2) FIG. 1 schematically shows possible ER-pathways. In particular, the figure shows that ER itself may be part of a multi-component transcription factor complex, wherein all of the proteins of this complex are potential targets for the methods of the invention as described herein. It is also clear that via PI3K another transcription factor complex may be formed which involves NFkappaB.

(3) FIG. 2 exemplarily shows a flow chart illustrating the choice of a suitable therapy depending on the results of a preferred method according to the invention.

(4) FIGS. 3 to 6 show results of IHC and PLA assays against ER and p300 (in this regard, cf. below, example 3).

DETAILED DESCRIPTION OF EMBODIMENTS

(5) As described above, the methods of the present invention allow for a reliable determination of the activity of certain pathways on protein levels. In particular, by using the proximity ligation assay it can be determined in which cells of a tissue sample the pathway is active. In this respect, it is also possible to provide evidence on the percentage of cancer cells in a tissue sample (as described above), preferably a tissue slide, which have the respective pathway activated. Furthermore, the methods described herein enable one to predict if certain (hormonal) therapies will be effective, whether resistance to (hormonal) treatment is expected or likely to develop, so that the matching therapy can be chosen depending on which pathway is found to be active.

(6) It is preferred that the methods according to the invention (as described above) are performed on tissue samples in vitro, preferably on tissue slides.

(7) The methods according to the invention can be advantageously combined with further, typically used stratification techniques. Such additional stratification tests can be made on the same tissue sample or on a different tissue sample obtained from the cancer patient. Furthermore, such addition stratification may be performed prior to the testing according to the invention, at the same time or later. For example, such additional testing can be performed by using IHC staining.

(8) With respect to the antibodies that may be used in connection with the proximity ligation assay(s) used in a method of the invention (as described herein), it is to be noted that different antibodies may be utilized which carry different labels in different color channels, so as to a testing for more than one pathway can be made in parallel on the same sample. Furthermore, it may be suitable to label certain antibodies, e.g. anti ER antibodies, with labels in different color channels, so as to e.g. IHC and PLA staining can be made with the same antibodies.

(9) As described above, particularly preferred methods according to one aspect of the present invention comprise the following step: (i) determining the activation status of an ER and/or PR signaling pathway by applying a Proximity Ligation Assay to detect in a tissue sample of the (cancer) patient the presence of at least one member of the ER family, said at least one member being part of a transcription factor complex, and at least one protein selected from the group consisting of TAFs, TBP, POLII, TFII, p300, CREP1, and CBP, wherein the at least one protein is part of the same transcription factor complex.

(10) Further preferred embodiments and features of methods according to the present invention are described above. Exemplary particularly preferred methods of the invention according to one aspect of the present invention comprise the following steps: (i) determining the activation status of an ER and/or PR signaling pathway by applying a Proximity Ligation Assay to detect in a tissue sample of the (cancer) patient the presence of at least one member of the ER family, preferably an ER dimer, said at least one member being part of a transcription factor complex, and at least one protein selected from the group consisting of TAFs, TBP, POLII, TFII, p300, CREP1, and CBP, preferably p300, wherein the at least one protein is part of the same transcription factor complex, and (v) determining the activation status of the PI3K pathway by applying a Proximity Ligation Assay to detect in the same tissue sample of the (cancer) patient an active PKB/Akt protein by measuring the phosphorylation status of PBK/Akt,
preferably wherein the patient is cancer patient, more preferably a breast cancer patient, preferably an ER and/or PR positive and HER2 negative breast cancer patient and,
respectively, the tissue sample is a tumor tissue sample from a breast cancer patient, preferably from an ER and/or PR positive and HER2 negative breast cancer patient.

(11) By employing such methods, basically four different test results are possible with four different treatment choices in an adjuvant setting (in all cases additional chemotherapy and radiation therapy are decided separately): 1. ER containing transcription factor complex (step (i)): positive pAkt (step (v)): negative .fwdarw. treatment choice: “endocrine treatment”. 2. ER containing transcription factor complex (step (i)): positive pAkt (step (v)): positive .fwdarw. treatment choice: “endocrine treatment” and (potential new) inhibitors of the PI3K pathway. 3. ER containing transcription factor complex (step (i)): negative pAkt (step (v)): negative .fwdarw. no endocrine treatment, no PI3K pathway drugs (potentially with exception of PI3K pathway inhibitors downstream of pAkt, such as TORC1 and/or TORC2 inhibitors). 4. ER containing transcription factor complex (step (i)): negative pAkt (step (v)): positive .fwdarw. (potential new) inhibitors of the PI3K pathway.

(12) It has to be noted that it is within the scope of the present invention to analyze the activity of two, three, more or all of the herein mentioned pathways (in this regard, cf. in particular steps (ii) to (vi) as described above), preferably by using the same tissue sample. This allows an improved choice of suitable treatment for the respective (cancer) patient.

(13) For example, depending on the outcome of testings performed on the basis of the herein described methods suitable therapeutics for (hormonal) treatment and/or, for example, suitable PI3K pathway inhibitors can be chosen from currently approved drugs (by e.g. FDA, CFDA, EMA, etc.) or from drugs in clinical trials (cf. e.g. FIG. 2 for drugs addressing PI3K pathway) or future drugs not yet known to the public.

(14) As described above, FIG. 1 schematically shows possible ER-pathways. In particular, the figure shows that ER itself may be part of a multi-component transcription factor complex, wherein all of the proteins of this complex are potential targets for the methods of the invention as described herein. In FIG. 1 the reference numbers have the following meaning:

(15) TABLE-US-00001 1 Estrogen 2 Estrogen Receptor 3 Growth Factors 4 Gene Expression 5 Antiapoptosis 6 Vasodilation 7 Dimerization 8 Nucleus

(16) As described above, FIG. 2 exemplarily shows a flow chart illustrating the choice of a suitable therapy depending on the results of a preferred method according to the invention. In FIG. 2 the reference numbers have the following meaning:

(17) TABLE-US-00002 1 Antibodies 3 ATP-competitive inhibitors 5 ATP-competitive inhibitors 7 ATP-competitive inhibitors 9 ATP-competitive & allosteric inhibitors 11 ATP-competitive & FKBP12-mediated inhibitors 51 Possible position(s) of intervention/mode of action of the following drugs (Pan-Pi3K): BKM120 (Novartis) XL-147 (Exelixis) PX-866 (Oncothyreon) GDC-0941 (Genentech) CH51327909 (Chugai Pharma) 53 Possible position(s) of intervention/mode of action of the following drugs (P110δ- specific): CAL-101 (Calistoga) 55 Possible position(s) of intervention/mode of action of the following drugs (P110α- specific): BYL719 (Novartis) GDC-0032 (Genentech) INK-1117 (Intellikine) 71 Possible position(s) of intervention/mode of action of the following drugs (PI3K/mTOR): PKI-587 (Pfizer) BEZ235 (Novartis) BGT226 (Novartis) PF-4691502 (Pfizer) GDC-0980 (Pfizer) XL-765 (Exelixis) SF1126 (Sernafor) GSK1059615 (GSK) 73 Possible position(s) of intervention/mode of action of the following drugs (TORC1/2): INK-128 (Intellikine) OSI-027 (Wyeth/Pfitzer) AZD-8055 (Astrazeneca) 91 Possible position(s) of intervention/mode of action of the following drugs (Akt): AZD5363 (Astrazeneca) GDC-0068 (Genentech) GSK690693 (GSK) VQD002 (Vloquest) 111 Possible position(s) of intervention/mode of action of the following drugs (TORC1): Everolimus/RAD001 (Novartis) Temsirolimus/CCI-779 (Wyeth/Pfitzer) Ridaforolimus/AP-23575 (Merck/Ariad)

(18) As described above, the present invention may be performed by the use of an automated equipment for performing the methods according to the invention, i.e. (a) for stratification of a patient, preferably a cancer patient, for assessing the suitability of a therapy for the patient, (b) for the prognosis of the outcome of a disease, preferably cancer, of a patient, preferably a cancer patient, or (c) for the prediction and/or detection of therapy resistance of a patient, preferably a cancer patient, towards (hormonal) therapy, as respectively described above.

(19) Preferably, the methods are thereby performed such that the readout is fluorescence.

(20) In general, the tests described herein in connection with the methods of the present invention can be performed manually, but it is also possible to use automated equipment, such as e.g. the benchmark of Ventana (Roche), thereby increasing reproducibility.

EXAMPLES

Example 1

(21) Indirect PLA assay to determine the activation status of an ER signaling pathway in cancer cell line samples spun on a substrate:

(22) Two antibodies raised in different species (e.g. mouse and rabbit) are used, e.g. mouse antibody against an epitope on the Estrogen-ER dimer complex (Monoclonal Mouse Anti-Human Estrogen Receptor α, clone 1D5, Dako/Cat #M7047) and rabbit antibody against p300 (Anti-p300 mouse anti-human mAb, clone NM-11, Millipore/Cat #NA46-100UG or Anti-p300 CT mouse anti-human mAb, clone RW 128, Millipore/Cat #05-257).

(23) TABLE-US-00003 Protocol: Deparaffinization: 3 × 10 min xylene, 40 min 5 min 100% EtOH, short 100% EtOH and 70% EtOH, tap water Antigen retrieval (optimum treatment 45 min to be established for every new antibody): 20-45 min 95° C., pH 6 or pH 9, cooling to RT, 10-20 min Wash 1-2× in PBS Blocking with Blocking Solution 30 min from the PLA kit: 1 drop per 1 cm2, incubation 30 min, 37° C., humidified Drip of Blocking Solution (do not wash) Incubation with 2 unlabelled primary 60 min antibodies from different species (e.g. mouse and rabbit), diluted in 0.1% BSA/PBS for 60 min, RT Wash 2 × 5 min in WashBuffer A 10 min Incubation with 2 PLA probes: e.g. 60 min PLUS-labeled-anti-mouse-antibody and MINUS-labeled-anti-rabbit- antibody in 1:5 in Antibody Diluent (AD) for 1 hr, 37° C., in the dark, humidified Wash 2 × 5 min in WashBuffer A 10 min (= 10 mM Tris/150 mM NaCl/0.05% Tween ™ 20) Incubation with Ligation mixture 1:5 30 min in MQ and 1:40 ligase for 30 min, 37° C., in the dark, humidified Wash 2 × 2 min in WashBuffer A  4 min Incubation with Amplification 100 min  Mixture 1:5 in MQ and 1:80 polymerase for 100 min, 37° C., in the dark, humidified (fluorescent probe included here as well) Wash 2 × 10 min in 1× WashBuffer B 20 min (= 200 mM Tris/100 mM NaCl) Wash 1 min in 0.01× WashBuffer B  1 min (= 2 mM Tris/1 mM NaCl) Let slide air-dry at RT in the dark 10 min Mounting with Duolink II Mounting 15 min Medium, wait at least 15 min before imaging Image analysis. (Number of PLA dots per cell or per cell compartment is counted either manually or software-aided.)

Example 2

(24) Direct PLA assay to detect phosphorylation status (=activation status) of Akt (to be made in addition to the assay of Example 1):

(25) Two antibodies are used (can be form the same species: e.g. mouse antibody against Akt1 (F-8L, Santa Cruz Biotech) and another mouse antibody against phospho-Akt (18F3.H11, Abcam, ab105731))

(26) TABLE-US-00004 Protocol: Deparaffinization: 3 × 10 min 40 xylene, 5 min 100% EtOH, short 100% EtOH and 70% EtOH, tap water Antigen retrieval (to be tested for 45 every new antibody): 20-45 min 95° C., pH 6 or pH 9, cooling to RT, 10-20 min Wash 1-2× in PBS Blocking with Blocking Solution 30 from the PLA kit: 1 drop per 1 cm2, incubation 30 min, 37° C., humidified Drip of Blocking Solution (do not wash) Incubation with 2 primary 60 antibodies (could be from same or different species) conjugated to 2 PLA probes (PLUS and MINUS oligonucleotides) in buffer PBS/0.1% BSA/1:20 Assay Reagent for 1 hr, RT, in the dark, humidified Wash 2 × 5 min in WashBuffer A 10 (= 10 mM Tris/150 mM NaCl/0.05% Tween ™ 20) Incubation with Ligation mixture 30 1:5 in MQ and 1:40 ligase for 30 min, 37° C., in the dark, humidified Wash 2 × 2 min in WashBuffer A 4 Incubation with Amplification 100 Mixture 1:5 in MQ and 1:80 polymerase for 100 min, 37° C., in the dark, humidified (fluorescent probe included here as well) Wash 2 × 10 min in 1× WashBufferB 20 (= 200 mM Tris/100 mM NaCl)

Example 3

3.1 IHC Assays for ER and p300 in Both ER+ (MCF7) and ER− (SKBR3) Cell Lines

(27) IHC assays for ER and p300 were first performed in both ER+ (MCF7) and ER− (SKBR3) cell lines.

(28) The analyses clearly showed that both ER and p300 are present in the nucleus of MCF7 cells in varying concentrations. In particular, in the original color images obtained from the stained IHC assay, a number of the cells were colored greenish, which indicates the presence of ER, a number of the cells were colored reddish, which indicates the presence of p300, and a number of the cells were colored yellowish, which indicates a combined signal (cf. FIG. 3, which shows a grayscale image that is based on an original color image obtained from the stained IHC assay; the reference numerals “ER”, “p300”, and “ER+p300” exemplarily point to cells indicates as ER, p300 or combined). The differences in color in the original color images (in FIG. 3 reduced to differences in brightness) indicate that the expressions and/or presence of both ER and p300 vary widely among the cells.

(29) In SKBR3 cells, in contrast, only p300 can be found. In fact, SKBR3 cells do not show any signal in the ER channel leaving the nuclei reddish colored by the anti-p300 antibody (cf. FIG. 4, which shows a grayscale image that is based on an original color image obtained from the stained IHC assay; the cells visible in the image are colored reddish in the original color images, indicating the presence of only p300).

3.2 PLAs Against ER and p300 in Both ER+ (MCF7) and ER− (SKBR3) Cell Lines

(30) PLAs against ER combined with p300 were performed in both ER+ (MCF7) and ER− (SKBR3) cell lines.

(31) The results clearly indicated the locations in the nuclei of cells where both factors are present in the same complex. In particular, in the original color images obtained from the stained PLAs, a high numbers of active transcription factor complexes are indicated for the MCF7 cells by the high number of reddish colored dots (cf. FIG. 5, which shows a grayscale image that is based on an original color image obtained from the stained PLA for the MCF7 cells; the bright spots are colored reddish in the original images, indicating active transcription factor complexes). As expected from the IHC assay, there is some variation among cells. As also expected, SKBR3 cells show hardly any active transcription factor complexes (cf. FIG. 6, which shows a grayscale image that is based on an original color image obtained from the stained PLA for the SKBR3 cells).

(32) Similar data are produced when performing a PLA against ER and the co-factor CREB (data not shown).

(33) While the invention has been illustrated and additionally described 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.

(34) 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.

(35) 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. The mere fact that test methods are recited in mutually different dependent claims does not indicate that a combination of these methods cannot be used to advantage.