JP-7855525-B2 - Histochemical systems and methods for evaluating the expression of EGFR and EGFR ligands in tumor samples

Inventors

• バーンズ， マイケル
• ブレッノ， ジョージ
• ケリー， ブライアン ディー．
• マーティン， ジム エフ．
• ムレイニー， アンドレア
• ピネダ， カルロス ティー．
• シャンムガム， カンダウェル

Assignees

• ヴェンタナ メディカル システムズ， インク．

Dates

Publication Date

20260508

Application Date

20210505

Priority Date

20200507

Claims (12)

1. (a) Contacting the tissue section with a human EGFR protein biomarker-specific binder and a detection reagent sufficient to directly or indirectly attach the first chromogen to the human EGFR protein biomarker-specific binder bound to the tissue section; (b) Contact the tissue section with an AREG protein biomarker-specific binder and a detection reagent sufficient to directly or indirectly attach a second chromogen to the AREG protein biomarker-specific binder bound to the tissue section; (c) Contacting the tissue section with an EREG protein biomarker-specific binder and a detection reagent sufficient to directly or indirectly attach a third chromogen to the EREG protein biomarker-specific binder bound to the tissue section; (d) Obtaining digital images of the tissue section containing the attached first, second, and third chromogens; (e) Identifying the region of interest (ROI) of the acquired digital image; (f) Identifying multiple object features within an identified ROI, wherein the object features are selected from at least biomarker-positive membrane staining, biomarker-positive cytoplasmic staining, and biomarker-positive cell staining; (g) (i) to obtain an ROI object metric by quantifying a plurality of identified object features, and (ii) to calculate a test object metric by dividing the obtained ROI object metric by an ROI metric , wherein the ROI metric is selected from the area of the identified ROI, the total number of cells in the identified ROI, the cell ratio in the identified ROI, or the ligand-receptor ratio in the identified ROI ; and (h) to calculate a score for acquired digital images based on the calculated test object metric using a scoring engine, wherein the scoring engine is derived from one or more models that correlate (i) training object metrics calculated from training samples from multiple patients with (ii) the respective clinical outcomes of multiple patients from which the multiple training samples originate; Includes, A method wherein a first pigment, a second pigment, and a third pigment have deconvolutionable colors.
2. The method according to claim 1, wherein the biomarker-specific conjugate is an antibody or an antigen-binding fragment thereof.
3. The method according to claim 1 or 2, wherein the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section.
4. The method according to any one of claims 1 to 3, wherein the tissue section is derived from a colorectal tumor sample.
5. The method according to any one of claims 1 to 3, wherein the tissue section is derived from a polyp.
6. The method according to any one of claims 1 to 3, wherein the tissue section is RAS wild-type.
7. The method according to any one of claims 1 to 6, wherein the tissue section does not contain a mutation that enables ligand-independent EGFR signaling.
8. The method according to any one of claims 1 to 7, wherein the tissue section does not contain a RAS protein having a mutation that confers resistance to EGFR monoclonal antibody therapy.
9. The method according to any one of claims 1 to 8, further comprising visualizing the chromogen using bright-field microscopy.
10. The method according to claim 1, further comprising evaluating, based on a calculated score, whether the patient from whom the tissue section was obtained is likely to benefit from anti-EGFR therapy.
11. The method according to claim 1, wherein the identified ROI is an EGFR-positive cell cluster.
12. The method according to claim 1, further comprising normalizing the test object metric before calculating the score.

Description

Cross-reference of related applications: This application claims priority and benefit of U.S. Provisional Patent Application No. US63/021,627, filed on 7 May 2020. Incorporation of Sequence Listings by Reference This application incorporates, by reference, the sequence listing file named 34457WO_SEQLIST_ST25, created on April 19, 2021, and with a size of 19,551 bytes, which was submitted together with this application in a computer-readable format. This invention relates to a histochemical method, system, and composition for evaluating the expression of human epidermal growth factor receptor (EGFR) protein and human EGFR ligand protein in colorectal tumors. Approximately 20% of colorectal cancer patients develop metastatic colorectal cancer (mCRC). More than half (50-60%) of these patients eventually develop an incurable, progressive disease, with a five-year survival rate of approximately 12.5%. In mCRC, two signaling pathways, the vascular endothelial growth factor receptor (VEGFR) pathway and the epidermal growth factor receptor (EGFR) pathway, are the focus of therapeutic development. Currently, the majority of mCRC patients receive cytotoxic chemotherapy in combination with either EGFR-targeted therapy or VEGF-targeted therapy. EGFR is overexpressed in approximately 70% of CRC cases and is associated with poor outcomes. EGFR-targeted inhibition with monoclonal antibodies such as cetuximab or panitumumab was approved by the FDA in 2004 and 2006 for the treatment of mCRC patients. These antibodies target the extracellular domain of EGFR, competing with endogenous ligands to prevent receptor activation. By inhibiting the EGFR signaling pathway, these biopharmaceuticals inhibit cell proliferation, differentiation, migration, and metastasis. Both drugs exhibit similar efficacy, with response rates of 10–15%. Reliable positive predictors regarding responsiveness to EGFR-directed therapy have long been lacking. Clinical trials have shown that EGFR inhibitors are most effective in patients lacking RAS pathway mutations. Point mutations in members of the RAS signaling pathway, such as KRAS, NRAS, and BRAF, result in persistent activation of downstream RAS-MAPK signaling, regardless of whether EGFR is pharmacologically inactivated. In addition to RAS and BRAF mutations, other alternative mechanisms, such as cMET or EGFR amplification, are involved in resistance to cetuximab or panitumumab. Mutations resulting in PI3K or PTEN loss (often occurring with RAS or BRAF mutations) may also be associated with a lack of response. In fact, mutations in RAS, BRAF, and PI3K account for over 60% of mCRC patients exhibiting de novo resistance to EGFR-targeted monoclonal antibodies. Of the 40% of patients with KRAS, NRAS, BRAF, and PI3K wild-type tumors (quadruple wild-type patients), approximately half (only 15%) benefited from anti-EGFR therapy, while over 20% were non-responders. See Perkins et al., Pharmacogenetics, Vol. 15, No. 7, pp. 1043–1052 (2014). Overexpression of EGFR ligands, including the ligands epiregulin (EREG) and amphiregulin (AREG), has been suggested as a predictor of anti-EGFR therapy. In one trial involving mCRC patients, the addition of anti-EGFR therapy increased survival in patients with high EREG expression levels from 5.1 months to 9.8 months compared to best supportive care alone. This result suggests that EGFR ligand expression may be a clinically useful biomarker for screening mCRC patients for EGFR inhibitor therapy. However, PCR-based detection systems cannot identify the spatial relationship between ligands and receptors. Immunohistochemical analysis of EGFR ligands has yielded varied results. For example, Khelwatty et al. (Oncotarget. 2017/1/31; 8(5):7666-7677) reported that co-expression of wild-type EGFR and at least one of its ligands (at a cutoff of EGFR-positive tumor cells > 5% and ligand staining intensity 2+) was significantly correlated with shorter progression-free survival and, consequently, a lower response rate to EGFR-targeted therapy. However, in their samples, EGFR staining was primarily observed in the cytoplasm, leading them to the theory that internal translocation of EGFR prevented EGFR therapy from inducing antibody-dependent cell-mediated cytotoxicity (ADCC). They further noted that up to 40% of the patients in their study may have previously received cetuximab therapy, which may have contributed to the downregulation of EGFR from the surface. Therefore, Khelwatty does not describe a clear correlation between the expression patterns of EGFR and EGFR ligands and the response to EGFR-targeted therapeutics. On the other hand, Yoshida et al. (Journal of Cancer Research and Clinical Oncology, March 2013, Vol. 139, No. 3, pp. 367-378) found a good correlation between four of the seven ligands (AREG, HB-EGF, TGFα, and EREG) and the clinical response to EGFR therapy, and that the response rate was significantly higher in patients expressing two or more of the four ligands. Howev