Treating bladder cancer patients and identifying bladder cancer patients responsive to treatment

Abstract

This document provides methods and materials involved in treating bladder cancer. This document also provides methods and materials involved in identifying bladder cancer patients likely or unlikely to respond to treatment (e.g., BCG therapy).

Claims

1. A method for treating bladder cancer, wherein said method comprises: (a) detecting the presence, in bladder cancer tissue of a bladder cancer patient not previously receiving BCG therapy to treat said bladder cancer, of a level of eosinophil infiltration or degranulation indicative of a decreased likelihood of responding to an anti-cancer immunotherapy, and (b) performing, to treat said bladder cancer, a radical cystectomy procedure on said patient who did not previously receive BCG therapy to treat said bladder cancer.

2. The method of claim 1, wherein said patient is a human patient.

3. The method of claim 1, wherein said method comprises detecting the presence, in bladder cancer tissue of said bladder cancer patient, of a ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells indicative of a decreased likelihood of responding to said anti-cancer immunotherapy.

4. The method of claim 3, wherein said ratio is a ratio of GATA-3.sup.+ to T-bet.sup.+ tumor-infiltrating T cells.

5. The method of claim 1, wherein said anti-cancer immunotherapy is BCG therapy.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. T cell subtype-specific antibodies identify unique T cell populations infiltrating Tis bladder tumors. Immunohistochemistry using antibodies specific for the master transcription factors, GATA-3 and T-bet, identify infiltrating Th2 polarized (brown staining cells) and Th1 polarized (red staining cells) lymphocytes, respectively. Scale bar=50 m.

(2) FIG. 2 is a graph plotting the relative number of bladder tumor infiltrating GATA-3.sup.+ (G) vs. T-bet.sup.+ (T) T cells (G/T ratio) at the time of initial biopsy/diagnosis. The G/T ratio differentiates Tis bladder cancer patients who are responsive from those who are un-responsive (refractory) to BCG immune therapy. G/T was scored for individual patient biopsies (n=9-10 per group) following immunohistochemical staining with antibodies specific for the respective transcription factors.

(3) FIG. 3 contains photographs of Tis bladder tumors stained with H&E or an eosinophil peroxidase-specific monoclonal antibody (EPX-mAb). Significant levels of eosinophil infiltration accompanied by activation (i.e., eosinophil degranulation) were observed. Immunohistochemistry using EPX-mAb demonstrates a robust tissue eosinophilia in Tis bladder tumors (red staining cells) that is often accompanied by eosinophil activation leading to areas of eosinophil peroxidase release/deposition (i.e., red staining areas of extracellular matrix). Scale bar=100 m.

(4) FIG. 4 is a graph plotting an the number of eosinophils or degranulating eosinophils present within tissue (EAI) for biopsies obtained at the time of initial diagnosis from Tis bladder cancer patients who were responsive or un-responsive (refractory) to BCG immune therapy. Biopsies from normal patients were used as a control. The EAI differentiated patients who are responsive from those who are un-responsive (refractory) to BCG immune therapy. EAI was scored for individual patient biopsies (n=9-10 per group) following immunohistochemical staining with the EPX-mAb.

(5) FIG. 5 is a graph plotting a Th2 biomarker signature for Tis bladder tumors from patients who were responsive or un-responsive (refractory) to BCG immune therapy. The Th2 biomarker signature represents a robust metric or score that stratifies bladder cancer patients into subgroups either responsive or refractory to BCG immune therapy. The numerical product of the signature derived from evaluations of the G/T ratio and EAI of a given bladder tumor (i.e., Th2 biomarker signature score) provided a quantifiable measure that was predictive of patient outcome following BCG immune therapy.

(6) FIG. 6 is a flow chart plotting possible bladder cancer treatments for bladder cancer patients identified as being likely or unlikely to respond to anti-cancer immunotherapy (e.g., BCG therapy).

(7) FIG. 7 is a flow chart plotting possible bladder cancer treatments for bladder cancer patients identified as being likely or unlikely to respond to anti-cancer immunotherapy (e.g., BCG therapy).

DETAILED DESCRIPTION

(8) This document provides methods and materials involved in treating bladder cancer. This document also provides methods and materials involved in identifying bladder cancer patients likely or unlikely to respond to treatment (e.g., BCG therapy). For example, this document provides methods and materials for using the ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells to identify bladder cancer patients (e.g., Tis bladder cancer patients) who are likely to respond to an anti-cancer immunotherapy. Examples of anti-cancer immunotherapy include, without limitation, BCG therapy or -interferon therapy. As described herein, patients having an initial bladder tissue biopsy with more tumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1 lymphoid cells can be identified or classified as being likely to respond to BCG therapy. Patients having an initial bladder tissue biopsy that does not have more tumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1 lymphoid cells can be identified or classified as being unlikely to respond to BCG therapy.

(9) A ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells can be determined using any appropriate method of assessing the number of Th2 lymphoid cells and Th1 lymphoid cells present within a tissue sample (e.g., a tumor biopsy). For example, immunohistochemistry, immunofluorescence, or FACS analyses techniques can be used to assess the level of tumor-infiltrating Th2 lymphoid cells and tumor-infiltrating Th1 lymphoid cells present within a tissue sample. When using immunohistochemistry techniques, antibodies to markers present on Th1 or Th2 lymphoid cells can be used. For example, antibodies such as anti-GATA-3 antibodies or anti-mouse/human GATA3 antibodies can be used to assess the number of Th2 lymphoid cells present within tissue, while antibodies such as anti-T-bet antibodies or anti-T-bet (e.g., H-210) antibodies can be used to assess the number of Th1 lymphoid cells present within tissue.

(10) Once the numbers of tumor-infiltrating Th1 and Th2 lymphoid cells are determined, they can be compared to each other or used to determine the ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells. If the patient is determined to have an initial bladder tissue biopsy with more tumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1 lymphoid cells, then that patient can be identified or classified as being likely to respond to BCG therapy. If the patient is determined to have an initial bladder tissue biopsy with less tumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1 lymphoid cells, then that patient can be identified or classified as being unlikely to respond to BCG therapy. In some cases, patients having a bladder biopsy with a ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells that is greater than or equal to 3 or a cut-off ratio can be identified or classified as being likely to respond to BCG therapy, while patients having a bladder biopsy with a ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells that is less than or equal to 1.5 or a cut-off ratio can be identified or classified as being unlikely to respond to BCG therapy. In some cases, a cut-off ratio can be 1.5, 2.0, 2.5, or 3.0. It is to be understood that a ratio of tumor-infiltrating Th1 lymphoid cells to tumor-infiltrating Th2 lymphoid cells can be used in a similar manner as described herein regarding a ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells by making appropriate adjustments to the calculations.

(11) Any appropriate sample can be used when measuring the levels of tumor-infiltrating Th1 and Th2 lymphoid cells. Such samples include, without limitation, tissue biopsies (e.g., bladder tissue biopsy) and tissue biopsy sections.

(12) This document also provides methods and materials involve in using the level of eosinophil infiltration and/or degranulation to identify bladder cancer patients (e.g., Tis bladder cancer patients) who are likely to respond to an anti-cancer immunotherapy (e.g., BCG therapy). As described herein, patients having an initial bladder tissue biopsy with elevated eosinophil infiltration and/or degranulation can be identified or classified as being likely to respond to BCG therapy. Patients having an initial bladder tissue biopsy that lacks elevated eosinophil infiltration and/or degranulation can be identified or classified as being unlikely to respond to BCG therapy.

(13) The level of eosinophil infiltration and/or degranulation present within a tissue sample (e.g., an initial bladder tissue biopsy) can be determined using any appropriate method. For example, immunohistochemistry, immunofluorescence, or FACS analyses techniques can be used to assess the level of eosinophil infiltration and/or degranulation present within a tissue sample. When using immunohistochemistry techniques, antibodies to eosinophils or components of eosinophils can be used. For example, antibodies such as anti-human eosinophil peroxidase antibodies and anti-mouse eosinophil peroxidase antibodies can be used to assess the number of eosinophils or degranulating eosinophils present within tissue.

(14) Once the numbers of eosinophils and/or degranulating eosinophils present within a tissue sample are determined, they can be compared to controls or used to calculate a score (e.g., an eosinophil activation index (EAI)). For example, an EAI can be calculated as described herein. In such cases, if the patient is determined to have an initial bladder tissue biopsy with an EAI greater than 4.0, 4.5, 5.0, or 5.5, then that patient can be identified or classified as being likely to respond to BCG therapy. If the patient is determined to have an initial bladder tissue biopsy with an EAI less than 5.5, 5.0, 4.5, or 4.0, then that patient can be identified or classified as being unlikely to respond to BCG therapy.

(15) Any appropriate sample can be used when assessing eosinophils and/or degranulating eosinophils as described herein. Such samples include, without limitation, tissue biopsies (e.g., bladder tissue biopsy) and tissue biopsy sections.

(16) In some cases, both the ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells and the level of eosinophil infiltration and/or degranulation can be used in combination to identify bladder cancer patients (e.g., Tis bladder cancer patients) who are likely to respond to an anti-cancer immunotherapy (e.g., BCG therapy). For example, the ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells can be multiplied or added to an EAI score to determine a Th2 biomarker signature that can be used to identify bladder cancer patients who are likely to respond to an anti-cancer immunotherapy (e.g., BCG therapy) as described herein.

(17) This document also provides kits that can be used to determine the levels of tumor-infiltrating Th1 and Th2 lymphoid cells and/or the levels of eosinophils and/or degranulating eosinophils. Such a kit can contain anti-GATA-3 antibodies and anti-T-bet antibodies. In some cases, a kit containing anti-GATA-3 antibodies and anti-T-bet antibodies can include anti-eosinophil peroxidase antibodies (e.g., anti-human eosinophil peroxidase antibodies). In some cases, a kit provided herein can contain a reference chart that indicates a reference level or cut-off ratio for the levels of tumor-infiltrating Th1 and Th2 lymphoid cells and/or the levels of eosinophils and/or degranulating eosinophils that are indicative of patient likely or unlikely to respond to an anti-cancer immunotherapy (e.g., BCG therapy) as described herein.

(18) This document also provides methods and materials for treating bladder cancer. For example, a bladder cancer patient can be assessed as described herein to determine whether the patient is likely or unlikely to respond to anti-cancer immunotherapy (e.g., BCG therapy). If the bladder cancer patient is identified as being likely to respond to anti-cancer immunotherapy (e.g., BCG therapy), then the patient can be administered an anti-cancer immunotherapy. For example, such a bladder cancer patient can be administered one or more (e.g., two, three, four, or more) rounds of BCG therapy. If the bladder cancer patient is identified as being unlikely to respond to anti-cancer immunotherapy (e.g., BCG therapy), then the patient can be administered an intravesical mitomycin C or thiotepa (N,NN-triethylenethiophosphoramide) chemotherapy, can be subjected to radical cystectomy, or both. For example, such a bladder cancer patient can be subjected to a radical cystectomy procedure to treat the bladder cancer. In some cases, a bladder cancer patient that is identified as being unlikely to respond to anti-cancer immunotherapy (e.g., BCG therapy) as described herein can be administered an intravesical mitomycin C or thiotepa chemotherapy, can be subjected to radical cystectomy, or both without having previously received an anti-cancer immunotherapy (e.g., BCG therapy) for the treatment of bladder cancer.

(19) In some cases, a bladder cancer patient can be assessed and treated as set forth in FIG. 6 or FIG. 7.

(20) The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1Assessing Bladder Cancer Outcomes

(21) A study was performed using initial biopsies of bladder cancer patients from the urogenital service. Briefly, immunohistochemistry was performed using commercially available antibodies specific for Th2 (GATA-3.sup.+; anti-human/mouse GATA3 (R&D Systems; Minneapolis, Minn.); and Th1 (T-bet.sup.+ anti-T-bet (H-210): sc-21003 (Santa Cruz Biotechnology, Santa Cruz, Calif.)) lymphocytes and a monoclonal antibody specific for human/mouse eosinophil peroxidase (EPX-mAb). See, e.g., Protheroe et al., Clin. Gastroenterol. Hepatol., 7:749-755 (2009).

(22) Biopsies from three subgroups of patients were used: (i) ten normal control subjects (i.e., patients whose initial biopsies were pathology-negative for bladder cancer; (ii) ten Tis bladder cancer patients unresponsive to BCG immune therapy (i.e., BCG refractory (BCG.sup.)); and (iii) ten Tis bladder cancer patients who displayed no evidence of tumor follow BCG immune therapy (i.e., BCG responsive patients (BCG.sup.+).

(23) The following was performed to determine the ratio of GATA-3.sup.+ tumor-infiltrating lymphoid cells to T-bet.sup.+ tumor-infiltrating lymphoid cells (i.e., G/T ratio). Briefly, tumor-infiltrating T cells were systematically assessed in individual Tis patient biopsies by pathologists. Intra/inter-observer reliabilities were evaluated by blinded assessments of the slides by other investigators. Six serial sections of each biopsy were taken for evaluation. Sections 1 of 6 and 6 of 6 were used as isotype negative controls for the immunohistochemical staining of the biopsy. Section 3 of 6 was stained with Hematoxylin and Eosin (H&E) for evaluation to identify the maximal focus of the mononuclear proinflammatory infiltration. Sections 2 of 6 and 4 of 6 were subjected to immunostaining using antibodies specific for GATA-3.sup.+ and T-bet.sup.+ tumor-infiltrating T cells, respectively. Ten random high powered (400) fields at the maximum focus of mononuclear infiltration were examined, and the numbers of GATA-3.sup.+ (G) and T-bet.sup.+ (T) tumor-infiltrating T cells were enumerated to define a G/T ratio for each patient sample. Representative photomicrographs of staining for each T cell-specific antibody are shown in FIG. 1. Evaluations of these sections revealed that it was possible to stratify bladder cancer patients on the basis of G/T ratio generating two subgroups, patients with a highly Th2 polarized immune microenvironment (G/T3) and patients with a Th1 polarized subgroup with a G/T ratio 1.5. More significantly, correlation of these scores with subsequent patient outcomes following BCG immune therapy demonstrated that patient responsiveness to BCG treatment (i.e., tumor elimination) trended with the higher G/T ratio patients (FIG. 2).

(24) EPX-mAb based immunohistochemistry was used to evaluate eosinophil infiltration of the bladder cancer patient biopsies as well as to quantify the level of degranulation observed in each biopsy (biopsy section 5 of 6). Representative examples of this EPX-mAb-based immunostaining are shown in FIG. 3. These evaluations led to the development of an eosinophil activation index (EAI) expressed as the product of a numerical score of eosinophil tissue infiltration (0=no eosinophils/40hpf; 1=1-4 eosinophils/40hpf; 2=5-10 eosinophils/40hpf; and 3=>10 eosinophils/40hpf) and a numerical score assessing eosinophil degranulation (1=no evidence of degranulation; 2=degranulation in <20% of the biopsy; 3=degranulation evident in 20-50% of the biopsy; and 4=degranulation evident in >50% of the biopsy). Eosinophil infiltration and degranulation were unique to bladder cancer with no evidence of either event in control (i.e., non-cancerous) bladder tissue. In addition, the initial biopsies of BCG responsive patients (BCG.sup.+) displayed a higher EAI relative to BCG refractory patients (BCG.sup.) (FIG. 4).

(25) The product of the G/T ratio and the EAI score from each patient (i.e., G/T ratio*EAI score resulted in a Th2 biomarker signature that easily stratified subjects into BCG responsiveness and unresponsive subgroups (FIG. 5).

(26) These results demonstrate that G/T ratios, eosinophil infiltration and/or degranulation, or both G/T ratios and eosinophil infiltration and/or degranulation can be used to assess bladder cancer biopsies (e.g., initial Tis bladder tumor biopsies) to determine whether or not the patient will be responsiveness to BCG immune therapy.

OTHER EMBODIMENTS

(27) It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.