WO2023222710A1 - Biomarqueur pour prédiction d'une réponse à l'immunothérapie par agoniste tlr 7 de lésions malpighiennes intraépithéliales et son utilisation thérapeutique - Google Patents
Biomarqueur pour prédiction d'une réponse à l'immunothérapie par agoniste tlr 7 de lésions malpighiennes intraépithéliales et son utilisation thérapeutique Download PDFInfo
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Classifications
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/57411—Specifically defined cancers of cervix
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to the field of medicine, in particular to the fields of oncology, immunology and immunotherapy of tumors. Specifically, the invention relates to immune-based biomarkers for predicting response to TLR 7-agonist immunotherapy of squamous intraepithelial lesions and the predictive and therapeutic use of these biomarkers.
- Cervical high-grade squamous intraepithelial lesion also referred to as cervical intraepithelial neoplasia (CIN) 2 and 3
- CIN cervical intraepithelial neoplasia
- cHSIL Cervical high-grade squamous intraepithelial lesion
- CIN cervical intraepithelial neoplasia
- hrHPV human papillomavirus
- LLETZ transformation zone
- surgical treatment strategies are ineffective in 10% of patients, failing to eradicate the hrHPV infection, resulting in often difficult to treat recurrences and repeated treatments or even hysterectomy.
- TLR7 tolllike receptor 7
- IFNy progen presenting cells
- TNFa tolllike receptor 7
- IL-12 progen presenting cells
- vHSIL vulvar HSIL
- TLR 7-agonist immunotherapy of squamous intraepithelial lesions and to provide for the prognostic and therapeutic use of these biomarkers.
- the invention relates to a method for predicting the response of a subject with a high-grade squamous intraepithelial lesion (HSIL) to toll-like receptor 7 (TLR7) agonist immunotherapy, wherein the method comprises the step of determining, in a pre-immunotherapy tissue sample of the lesion, a count of at least one of intraepithelial cells and stromal cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c, and wherein: a) the subject is predicted to be a complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the markers CD4, CD68 and CD11 c is higher than a cut-off value; or b) the subject is predicted to be a noncomplete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and strom
- the count of cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is corrected for a count of cells per mm 2 that are positive for at least one of the markers FOXP3 and CD163.
- the cut-off value is determined using a receiver operating characteristic (ROC) curve of the counts of at least one of intraepithelial cells and stromal cells that are positive for at least one of the markers CD4, CD68 and CD11 c in pre-immunotherapy biopsies of the lesions of complete responders and non-complete responders in a validation cohort, wherein preferably, the validation cohort comprises at least 10, 20, 35, 50, 75, 100, 150, 300, 600 or 1000 HSIL patients.
- the ROC curve for the cut-off value has an area-under- the-curve (AUC) of more than 0.7, 0.75, 0.80, 0.85, 0.90 or 0.95.
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is or is higher than a cut-off value in the range of 90 - 360 cells/mm 2 , wherein preferably the cut-off value is or is higher than 178.5 cells/mm 2 .
- the cut-off value for the pre-immunotherapy count of stromal cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is or is higher than a cut-off value in the range of 750 - 1500 cells/mm 2 , wherein preferably the cut-off value is higher than 1187 cells/mm 2 .
- the count of cells that are positive for at least one of the markers CD4, CD68 and CD11 c, and the count of cells that are positive for at least one of the markers FOXP3 and CD163, are determined by immunostaining, wherein preferably, the immunostaining for the markers FOXP3 and CD163 is different than the staining for the markers CD4, CD68 and CD11 c and wherein more preferably, the immunostaining is performed by at least one of immunohistochemistry and immunofluorescence.
- At least one of: i) the count cells positive for the markers CD4, CD68 and CD11 c is determined by immunostaining in the same color (a first color); and, ii) the count of cells positive for the markers FOXP3 and CD163 is determined by immunostaining in the same color (a second color), and wherein preferably, the immunostaining of the cells positive for the markers FOXP3 and CD163 is performed using a different color than the immunostaining of the cells positive for the markers CD4, CD68 and CD11 c (i.e. the first and second colors are different colors).
- the invention relates to a method for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy, wherein the method comprises the step of determining, in a pre-immunotherapy tissue sample of the lesion, a count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + , and wherein: a) the subject is predicted to be a complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + is higher than a cut-off value; or b) the subject is predicted to be a non-complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepitheli
- the cut-off value is determined using a receiver operating characteristic (ROC) curve of the counts of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ and CD68 + CD163 _ in preimmunotherapy biopsies of the lesions of complete responders and non-complete responders in a validation cohort, wherein preferably, the validation cohort comprises at least 10, 20, 35, 50, 75, 100, 150, 300, 600 or 1000 HSIL patients.
- the ROC curve for the cutoff value has an area-under-the-curve (AUC) of more than 0.7, 0.75, 0.80, 0.85, 0.90 or 0.95.
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than a cut-off value in the range of 10 - 40 cells/mm 2 , wherein preferably, the cut-off value is or is higher than 20 cells/mm 2 .
- the cut-off value for the pre-immunotherapy count of stromal cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than a cut-off value in the range of 85 - 340 cells/mm 2 , wherein preferably, the cut-off value is or is higher than 170 cells/mm 2 .
- the counts of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ and CD68 + CD163 _ in the pre-immunotherapy biopsies of the lesion is determined by multispectral immunofluorescence on the tissue sample.
- the HSIL can be a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL, whereby preferably the HSIL is a cervical HSIL.
- the immunotherapy can be local immunotherapy
- the TLR7 agonist can be at least one of imiquimod and resiquimod.
- the invention pertains to a TLR7 agonist for use in immunotherapy of an HSIL in a subject wherein the subject is predicted to be a complete responder in any of above methods of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy.
- the HSIL is a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL, wherein preferably the HSIL is a cervical HSIL.
- the immunotherapy is local immunotherapy.
- the TLR7 agonist is at least one of imiquimod and resiquimod.
- the invention pertains to a method for treating an HSIL in a subject, the method comprising predicting the subject to be a complete responder or a non-complete responder in any of above methods of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy, and i) treating the subject with TLR7 agonist immunotherapy, if the subject is predicted to be a complete responder, by administering a TLR7 agonist to the subject’s HSIL; or ii) treating the subject with HSIL excision therapy if the subject is predicted to be a noncomplete responder, wherein preferably the HSIL excision therapy comprises surgical large loop excision of the transformation zone (LLETZ).
- LLETZ transformation zone
- the HSIL is a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL, wherein preferably the HSIL is a cervical HSIL.
- the immunotherapy is local immunotherapy.
- the TLR7 agonist is at least one of imiquimod and resiquimod.
- invention in a fourth aspect, relates to kits for performing any of above methods of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy.
- a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.
- biomarker refers to an epitope, antigen or receptor that is expressed on immune cells or different subsets of of immune cells. Expression of some biomarkers is specific for cells of a particular lymphoid or myeloid lineage or maturational pathway, and the expression of others varies according to the state of activation, position, or differentiation of the same cells.
- a biomarker may be a cell surface biomarker or an intracellular biomarker. In one embodiment, the biomarkers used in the methods disclosed herein are all cell surface biomarkers. In another embodiment, the biomarkers used in the methods disclosed herein are all intracellular biomarkers.
- the biomarkers used in the methods disclosed herein include both cell surface biomarkers and intracellular biomarkers.
- Exemplary biomarkers include, without limitation, a CD4 biomarker, a CD68 biomarker, a CD11 c biomarker, a FOXP3 biomarker, CD163 biomarker, a CD3 biomarker and a CD8 biomarker.
- immunohistochemistry and “immunofluorescence” refer to a method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent, such as an antibody.
- a sample is contacted with an antibody under conditions permitting antibody-antigen binding.
- Antibody-antigen binding can be detected by means of a detectable label (detectable e.g. by color or fluorescence) conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which binds specifically to the primary antibody (indirect detection).
- a cell is counted as positive for at least one of a plurality of markers, e.g. A, B and C, when expression of at least one of those markers, e.g. A, is detected on or in a given cell, whereby the markers can be detected in a method as defined herein. That cell need not express each of the markers A, B and C, as detection of only one of the markers already qualifies the cell as positive for at least one of the markers A, B and C. However, it is also understood that a cell that is positive for more than one of the markers A, B and C is only counted once in the count of cells that are positive for at least one of the markers A, B and C.
- the term “cut-off value” refers to a dividing point on measuring scales where test results are divided into different categories.
- the subject when the sample score is greater than the cut-off score, the subject is predicted to be a complete responder to TLR7 agonist immunotherapy, and vice versa, the subject is predicted to be a non-complete responder to TLR7 agonist immunotherapy when the sample score is not greater than the cut-off score.
- the cut-off value may be determined based on the performance of the prediction model.
- a receiver operating characteristic (ROC) curve may be used to evaluate the performance of the prediction model. The ROC curve may illustrate the prognostic ability of the prediction model as its cut-off value is varied.
- the ROC curve is usually generated by plotting the sensitivity against the specificity.
- An area-under-the-curve (AUC) may be determined based on the ROC curve.
- the AUC may indicate the probability that a classifier (i.e., the prediction model) will rank a randomly chosen positive instance higher than a randomly chosen negative one.
- the inventors performed a comprehensive in-depth characterization of the immune microenvironment using multispectral immunofluorescence on a cohort of cHSIL patients treated with topical imiquimod, investigating both pre- and on treatment cHSIL tissue, in the context of their clinical response. They show that imiquimod has the strongest effects on immune cell composition in lesions with high pre-existing immunity.
- imiquimod has the strongest effects on immune cell composition in lesions with high pre-existing immunity.
- a strong pre-existing infiltration by CD3 + CD8 FOXP3 _ T cells, CD68 + CD163 _ M1-like macrophages and CD11 c + dendritic cells accurately distinguished complete responders from non-responders.
- the invention relates to a method for predicting the response of a subject with a squamous intraepithelial lesion to toll-like receptor 7 (TLR7) agonist immunotherapy.
- the squamous intraepithelial lesion is a high-grade squamous intraepithelial lesion (HSIL).
- the method preferably comprises the step of determining a pre-immunotherapy count of at least one of T cells, macrophages and dendritic cells in tissue of the lesion, preferably in a preimmunotherapy biopsy of the lesion.
- the method comprises the step of determining a pre-immunotherapy count of at least one of CD4 + T cells, CD68 + macrophages and CD11 c + dendritic cells in tissue of the lesion, preferably in a pre-immunotherapy biopsy of the lesion.
- the method comprises the step of determining a count of cells in a pre-immunotherapy tissue of the lesion, wherein the counted cells are defined by the combinations of markers or phenotypes as defined hereinbelow.
- the count is preferably a count of cells per volume unit of the tissue, or in case of sections, e.g. thin sections (e.g. of formalin-fixed paraffin-embedded sections), a count of cells per surface unit, e.g. mm 2 , of the section of the tissue, or other types of units such as count per high powerfield or percentage of pixels in the image.
- the count is performed on a pre-immunotherapy tissue sample of the lesion, preferably a biopsy of the lesion.
- a pre-immunotherapy tissue sample or biopsy of the lesion is herein understood as tissue from the lesion that is taken prior to that the TLR7 agonist immunotherapy of the lesion has commenced. Since the count is performed ex vivo, on tissue from the lesion (e.g. a biopsy), a method of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy is thus an in vitro method.
- the count comprises at least one of intraepithelial cells and stromal cells. In one embodiment, the count comprises at least intraepithelial cells.
- the count of cells that are positive for combinations of markers as defined herein are determined by immunostaining.
- the immunostaining is performed by at least one of immunohistochemistry and immunofluorescence, of which immunohistochemistry is preferred.
- the immunostaining is performed on a tissue section of the lesion, preferably on a section of formalin-fixed paraffin-embedded (FFPE) tissue material of the lesion. Sectioning of the paraffin-embedded tissue is widely used and well-known in the art of histology and pathology.
- the tissue material is a biopsy of a histologically confirmed HSIL.
- the method comprises the step of determining the count of cells that are positive for at least one of the markers CD4, CD68 and CD11 c.
- the method comprises the step of determining, in a pre-immunotherapy tissue sample of the lesion, the count of at least one of intraepithelial cells and stromal cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c.
- the method comprises counting the cells that are positive for the markers CD4, CD68 and CD11 c.
- the count of cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is corrected for a count of cells per mm 2 that are positive for at least one of the markers FOXP3 and CD163.
- the count of cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c can thus refer to the count of cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c as such, but preferably refers to the count of cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD1 1 c is corrected for a count of cells per mm 2 that are positive for at least one of the markers FOXP3 and CD163.
- the subject is predicted to be a complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the markers CD4, CD68 and CD11 c is higher than a cut-off value; or, the subject is predicted to be a non-complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the markers CD4, CD68 and CD11 c is not higher than the cut-off value.
- the cut-off value of the counts of cells having the above markers may be determined based on the performance of the prediction model.
- a receiver operating characteristic (ROC) curve may be used to evaluate the performance of the prediction model.
- the cut-off value is determined using a receiver operating characteristic (ROC) curve of the counts of at least one of intraepithelial cells and stromal cells that are positive for at least one of the markers CD4, CD68 and CD11 c in pre-immunotherapy biopsies of the lesions of complete responders and non-complete responders in a validation cohort, wherein preferably, the validation cohort comprises at least 10, 20, 35, 50, 75, 100, 150, 300, 600 or 1000 HSIL patients.
- the ROC curve for the cut-off value has an area-under-the-curve (AUC) of more than 0.7, 0.75, 0.80, 0.85, 0.90 or 0.95.
- the sensitivity of the prediction model for the response of a subject with an HSIL to TLR7 agonist immunotherapy is equal to or greater than 70, 75, 80, 90 or 95%.
- the specificity of the prediction model for the response of a subject with an HSIL to TLR7 agonist immunotherapy is equal to or greater than 70, 75, 80, 90 or 95%.
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is or is higher than 90, 90.5, 91 , 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 100, 100.5, 101 , 101 .5, 102, 102.5, 103, 103.5,
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c (without correction for the markers FOXP3 and CD163) is or is higher than 178.5 cells/mm 2 .
- the cut-off value for the pre-immunotherapy count of stromal cells per mm 2 that are positive for at least one of the markers CD4, CD68 and CD11 c is or is higher than 750, 751 , 752, 753, 754, 755, 756, 757, 758, 759, 760, 761 , 762, 763, 764, 765, 766, 767, 768, 769, 770, 771 , 772, 773, 774, 775, 776, 777,
- the immunostaining for the markers FOXP3 and CD163 is different than the immunostaining for the markers CD4, CD68 and CD11 c. In one embodiment, at least one of: i) the count cells positive for the markers CD4, CD68 and CD11 c is determined by immunostaining in the same color; and, ii) the count of cells positive for the markers FOXP3 and CD163 is determined by immunostaining in the same color, whereby preferably, the immunostaining of the cells positive for the markers FOXP3 and CD163 is performed using a different color than the immunostaining of the cells positive for the markers CD4, CD68 and CD11 c. In one embodiment, the cells positive for the markers FOXP3 and CD163 are identified by Vector Red staining and the cells positive for the markers CD4, CD68 and CD11 c are identified by DAB staining.
- no further membranous and/or nuclear staining is applied.
- the method of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy comprises the step of determining, in a pre- immunotherapy tissue sample (e.g. a biopsy) of the lesion, a count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ CD68 + CD163 _ and CD11 c + .
- a pre- immunotherapy tissue sample e.g. a biopsy
- the subject is predicted to be a complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + is higher than a cut-off value; or the subject is predicted to be a non-complete responder to TLR7 agonist immunotherapy if the subject’s count of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + is not higher than the cut-off value.
- the count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + is corrected for a count of cells per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + (of regulatory T cells).
- the count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + can thus refer to the count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + as such, but can also refers to the count of cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + , corrected for a count of cells per mm 2 that are positive for at least one of the phenotype CD3 + CD8 FOXP3 + .
- the cut-off value for the counts of cells having the above phenotypes may be determined based on the performance of the prediction model.
- a receiver operating characteristic (ROC) curve may be used to evaluate the performance of the prediction model.
- the cut-off value is determined using a receiver operating characteristic (ROC) curve of the counts of at least one of intraepithelial cells and stromal cells that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + in pre-immunotherapy biopsies of the lesions of complete responders and non-complete responders in a validation cohort, wherein preferably, the validation cohort comprises at least 10, 20, 35, 50, 75, 100, 150, 300, 600 or 1000 HSIL patients.
- ROC receiver operating characteristic
- the ROC curve for the cut-off value has an area-under-the-curve (AUC) of more than 0.7, 0.75, 0.80, 0.85, 0.90 or 0.95.
- AUC area-under-the-curve
- the sensitivity of the prediction model for the response of a subject with an HSIL to TLR7 agonist immunotherapy is equal to or greater than 70, 75, 80, 90 or 95%.
- the specificity of the prediction model for the response of a subject with an HSIL to TLR7 agonist immunotherapy is equal to or greater than 70, 75, 80, 90 or 95%.
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21 , 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31 , 31 .5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5 or 40 cells/mm 2 .
- the cut-off value for the pre-immunotherapy count of intraepithelial cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than 20 cells/mm 2 .
- the cut-off value for the pre-immunotherapy count of stromal cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91 , 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 100, 100.5, 101 , 101.5, 102, 102.5, 103,
- the cut-off value for the pre-immunotherapy count of stromal cells per mm 2 that are positive for at least one of the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ and CD11 c + minus the count of per mm 2 that are positive for the phenotype CD3 + CD8 FOXP3 + is or is higher than 170 cells/mm 2 .
- the counts of at least one of intraepithelial cells and stromal cells that are positive for the phenotypes CD3 + CD8 FOXP3 _ , CD68 + CD163 _ or CD3 + CD8 FOXP3 + is determined by multispectral immunofluorescence on the tissue by methods known in the art per se.
- the HSIL is a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL. In a preferred embodiment, the HSIL is a cervical HSIL.
- the immunotherapy is local immunotherapy.
- the TLR7 agonist is at least one of imiquimod and resiquimod.
- the invention pertains to therapeutic methods based on the above methods of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy.
- the invention pertains a TLR7 agonist for use in immunotherapy of an HSIL in a subject, wherein the subject is predicted to be a complete responder in a methods of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy as defined herein above.
- the TLR7 agonist is for use in immunotherapy of an HSIL in a subject, wherein the HSIL is a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL. In a preferred embodiment, the HSIL is a cervical HSIL.
- the TLR7 agonist is for use in immunotherapy of an HSIL in a subject, wherein the immunotherapy is local immunotherapy.
- the TLR7 agonist is for use in immunotherapy of an HSIL in a subject, wherein the TLR7 agonist is at least one of imiquimod and resiquimod.
- the invention pertains to a method of treating an HSIL in a subject, the method comprising predicting the subject to be a complete responder or a non-complete responder in a method for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy as herein defined above, and i) treating the subject with TLR7 agonist immunotherapy, if the subject is predicted to be a complete responder, by administering a TLR7 agonist to the subject’s HSIL; or ii) treating the subject with HSIL excision therapy if the subject is predicted to be a non-complete responder, wherein preferably the HSIL excision therapy comprises surgical large loop excision of the transformation zone (LLETZ).
- the HSIL excision therapy can comprise hysterectomy.
- the HSIL is a cervical, vulvar, vaginal, penile, anal or peri-anal HSIL. In a preferred embodiment, the HSIL is a cervical HSIL.
- the immunotherapy is local immunotherapy.
- the TLR7 agonist is at least one of imiquimod and resiquimod.
- the invention in a third aspect, relates to a kit for use in a method of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy.
- the kit therefore preferably comprises reagents for the immunostaining of the markers defined above and/or for the immunostaining of the markers of the phenotypes as defined above.
- Such reagents at least include ligands that bind the markers, such as antibodies that specifically bind the markers.
- the kit thus comprises a combination of ligands that specifically bind at least one of the markers CD4, CD68 and CD11 c. In one embodiment, the kit comprises a combination of ligands that specifically bind the markers CD4, CD68 and CD11 c. In one embodiment, the kit further comprises a combination of ligands that specifically bind at least one of the markers FOXP3 and CD163. In one embodiment, the kit further comprises a combination of ligands that specifically bind the markers FOXP3 and CD163. In one embodiment, the kit thus comprises a combination of ligands that specifically bind the markers CD4, CD68, CD11 c, FOXP3 and CD163. In a preferred embodiment the ligands are antibodies.
- the ligands in the kit that specifically bind the markers CD4, CD68 and CD11 c are detectable with a first common secondary ligand, e.g. antibodies of a first species.
- the ligands in the kit that specifically bind the markers FOXP3 and CD163 are detectable with a second common secondary ligand, e.g. antibodies of a second species.
- the antibodies of the first species can e.g. be rabbit IgG antibodies and the antibodies of the second species mouse IgG antibodies, or vice versa.
- the kit thus further comprises the secondary ligands as defined above.
- the kit comprises a combination of ligands that specifically bind at least one of the markers CD3, CD8, FOXP3, CD68, CD163 and CD11 c. In one embodiment, the kit comprises a combination of ligands that specifically bind the markers CD3, CD8, FOXP3, CD68, CD163 and CD11 c. In a preferred embodiment the ligands are antibodies. The ligand can be directly labelled with a fluorochrome and/or can be detected indirectly using a secondary ligand labelled with a fluorochrome.
- each ligand that specifically bind one of the markers CD3, CD8, FOXP3, CD68, CD163 and CD11 c is labelled by a different fluorochrome, be it by direct labelling or through secondary ligand labelled with a fluorochrome, thus allowing multispectral immunofluorescence analysis to establish whether a given cell (in tissue, e.g. a biopsy of an HSIL lesion) has a phenotype selected from the group consisting of: CD3 + CD8 FOXP3 _ , CD68 + CD163 _ , CD3 + CD8 FOXP3 + and CD11 c + .
- the kit further comprises a manual with instruction for performing a method of the invention for predicting the response of a subject with an HSIL to TLR7 agonist immunotherapy.
- A) Multispectral immunofluorescence T cell panel depicting the full seven color panel on a cHSIL biopsy, including all individual markers (DAPI, CD3, CD8, FOXP3, PD1 , TIM3, Tbet).
- B) Multispectral immunofluorescence myeloid cell panel depicting the full seven color panel on a cHSIL biopsy, including all individual markers (DAPI, CD68, CD163, CD14, CD1 1 c, PDL1 , CD33).
- C) Statistically significant changes (Wilcoxon paired signed rank test) in immune cell infiltration upon imiquimod treatment in cHSIL’s epithelium and stroma (n 35), as measured pre-treatment and after 10 weeks of imiquimod treatment.
- a red interaction indicates that these two cell phenotypes frequently interact in NR lesions (more than expected based on chance, Z-score>2), and are spatially separated in CR lesions (Z-score ⁇ - 2).
- a green interaction indicates that these two cell phenotypes frequently interact in CR lesions (more than expected based on chance, Z-score>2), and are spatially separated in NR lesions (Z- score ⁇ -2).
- the biomarker was calculated as the sum of all cells with a completely brown membrane and unstained nucleus (CD4 + /CD68 + /CD11 c + FOXP3- CD163 ) minus all cells with a brown membrane and red nucleus (CD4 + FOXP3 + ), for both the B) epithelium and C) stroma.
- Simplified epithelial immune biomarker is excellent CHSIL immune biomarker for imiquimod (CIBI).
- A) Multispectral immunofluorescence biomarker performance, consisting of the sum of total of CD4 + , CD68 + CD163 _ and CD11 c + cells minus the total of FOXP3 + cells in the epithelium as determined by the receiver operating characteristic (ROC) curve (n 32), and the same in B) for the dual immunohistochemical biomarker.
- FFPE formalin-fixed paraffin-embedded
- Biopsies were taken at three time points: pre-imiquimod (diagnostic), on imiquimod treatment (10 weeks) and after imiquimod treatment (20 weeks).
- Persistent HPV + cHSIL (CIN2-3) at 20 weeks was defined as non-responsiveness (NR) and was an indication for LLETZ. All patients with LSIL (CIN1) or no dysplasia at 20 weeks underwent 6 months of follow-up, after which a PAP smear was taken.
- Complete response (CR) was defined as cytologically confirmed lesion clearance at 6 months (PAP1), and partial response (PR) was defined as >PAP1 at 6 months.
- An overview of the study workflow is presented in Supplemental Figure 1.
- Two previously developed seven-color multispectral immunofluorescence panels were applied, one for T cells, consisting of CD3, CD8, FOXP3, TIM3, Tbet, PD-1 , DAPI, and one for myeloid cells, consisting of CD14, CD33, CD68, CD1 1 c, CD163, PD-L1 , DAPI. 8
- a combination of direct detection (primary antibody directly labelled with fluorochrome) and indirect detection (fluorochrome labelled secondary antibody) of markers was used, and dim markers (PD- L1 , PD-1 and Tbet) were tyramide signal amplified with Opal (PerkinElmer) to enable their detection by fluorescence microscopy.
- HRP horseradish peroxidase
- AP alkaline phosphatase
- DAB oxidized by HRP to a brown chromogen
- Vector Red oxidized by AP to a red chromogen
- TAE tumor microenvironment
- Immune cell counts were normalized for tissue size (cells/mm 2 epithelium and cells/mm 2 stroma). A threshold of a median cell count >10 cells/mm 2 in at least one response group and at least one time point was applied to study biologically common phenotypes.
- Imiquimod induces a strong intraepithelial influx by T cells and a decrease in macrophages.
- Imiquimod increased the numbers of epithelial CD3 + CD8 FOXP3 _ T cells and stromal CD14 + CD68 CD11 c monocytes, albeit that this varied per patient.
- stromal M1-like (CD68 + CD163 ) and epithelial and stromal M2-like (CD68 + CD163 + ) macrophages was observed in almost all patients upon imiquimod treatment (Figure 1C, Supplemental Figure 2).
- Six months after imiquimod treatment 21 patients had a complete response (CR), 3 patients a partial response (PR) and 11 patients were non-responders (NR).
- the number of total T cells did not differ between CR and NR, but clearly the total numbers of intraepithelial and stromal CD3 + CD8 FOXP3 _ T cells were higher in CR patients, while the intraepithelial and stromal numbers of CD3 + CD8 FOXP3 + regulatory T cells were higher in NR patients ( Figure 2C, Supplemental Figure 4A).
- the median myeloid cell counts were more than two-fold higher in CR compared to NR ( Figure 2A), with especially more intraepithelial and stromal presence of M1-like macrophages and CD68 CD14 CD1 1 c + dendritic cells in CR patients ( Figure 2C).
- This study is the first to identify a strong and easily applicable predictive biomarker for the clinical response of cHSIL patients to local immunotherapy with imiquimod, the CIBI.
- This biomarker is grounded on a comprehensive in-depth study of the phenotype and spatial composition of immune cells present in cHSIL, showing a strong relation between complete response to therapy and pre-existing CD4 + T cell, M1 -like macrophage and DC infiltration.
- These data formed the basis for a simple specific and sensitive single color immunohistochemical detection and scoring method with a positive and negative predictive value >90%, thereby adding significant value to the current ⁇ 60% a priori chance of clinical response of cHSIL to imiquimod.
- CIBI may be of great clinical value in the selection of patients responsive to imiquimod, hereby preventing unnecessary exposure to adverse effects of the intensive imiquimod treatment regimen.
- CIBI-based personalized therapy will improve therapy efficacy in the selected cHSIL patients, can be used to motivate therapy-adherence, and may prevent surgical LLETZ treatment, thereby reducing the risk of potential future obstetric complications (i.e. cervical insufficiency and subsequent premature deliveries).
- the immune composition of cHSIL in patients with spontaneous regression includes all actors of the immune response required to mediate lesion clearance, and this can be observed in about 20% of cHSIL patients.
- 13 14 Spontaneous cHSIL regression is associated with a higher CD8 + (granzyme B + ) and CD4 + T cell infiltrate, and low infiltration by CD25 + regulatory T cells and CD138 + B cells, when compared to lesions that persisted.
- 13-17 Spontaneous cLSIL regression is associated with reduced CD68 + macrophage infiltration compared to lesions that persisted. 18
- high numbers of CD4 + T cells, Tbet + T cells and CD11 c + DCs are associated with the absence of recurrences after surgical therapy.
- CD8 + T cells Cell types that were present in low numbers in CR before treatment and did not increase upon imiquimod treatment, i.e. CD8 + T cells, are not likely to form key mediators of lesion clearance.
- CR complete response
- PR partial response
- NR no response
- CIN cervical intraepithelial neoplasia
- PAP Papanicolaou dysplasia classification for cervix smear cytology
- x sample not available. Supplemental Table 1. Panel designs.
- Hendriks K e.a. Topical Imiquimod treatment of high-grade Cervical Intraepithelial Neoplasia (TOPIC-3): a non-randomized multicentre study. Journal of Immunotherapy 2022
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Abstract
La présente invention concerne des procédés de prédiction de la réponse d'un sujet avec une lésions malpighiennes intraépithéliales de haut grade (HSIL) à une immunothérapie par agoniste du récepteur de type Toll 7 (TLR7). Plus particulièrement, les procédés de prédiction de la réponse à l'immunothérapie par agoniste du TLR7 consistent à déterminer, dans un échantillon de tissu de la lésion préimmunothérapie, le nombre de cellules intraépithéliales et/ou stromales qui sont positives pour les biomarqueurs CD4, CD68 et CD11c, ou les cellules ayant les phénotypes CD3+CD8- FOXP3-, CD68+CD163- et CD11c+, de préférence corrigées pour les cellules ayant un phénotype CD3+CD8- FOXP3+. L'invention concerne également des agonistes TLR7 destinés à être utilisés dans l'immunothérapie contre la HSIL chez des sujets chez qui l'on prédit une réponse complète selon les procédés de prédiction de l'invention.
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