Pten clone 6h2

AR-v7 splice variant status (positive or negative) was assessed in baseline plasma exosome mRNA, by quantitative PCR (qPCR; ref. 14 (link)), validated in the Clinical Diagnostics Laboratory (Eli Lilly and Company) prior to use.
To define the patient population with PTEN loss tumors in the exploratory analyses, PTEN status was assessed by an IHC assay performed on archival soft tumor tissues at Neogenomics, using a validated assay. PTEN homolog protein expression was determined on formalin-fixed and paraffin-embedded sections using PTEN, Clone 6H2.1 (Dako). A qualified pathologist evaluated results according to prespecified interpretation guideline: a negative PTEN specimen was defined as having <5% of cells that exhibited staining in nuclear and/or cytoplasmic pattern at any intensity and a positive PTEN specimen as having ≥5% of cells that exhibited staining in nuclear and/or cytoplasmic pattern at any intensity. The assay was not validated for bone metastases due to the need for a decalcification step and only soft tissue samples were used. The analysis was conducted prior to unblinding. rPFS by PTEN status was examined.

Immunohistochemical staining of mismatch repair (MMR)‐related proteins (MLH1, MSH2, MSH6, and PMS2) and PTEN was performed at the Department of Pathology, Tohoku University Hospital, Japan, using the following antibodies: an anti‐human mouse MLH‐1 clone G168‐15 (1/100; BD Biosciences, San Jose, CA, USA), an anti‐MSH2 mouse mAb clone FE11 (1/200; Sigma‐Aldrich, St. Louis, MO, USA), an anti‐MSH6 mouse clone 44 (1/1000; BD Biosciences), an anti‐PMS2 mouse clone 16‐4 (1/100; BD Biosciences), and a PTEN clone 6H2.1 (1/100; DAKO, Carpinteria, CA, USA). Cases in which nuclei were stained were deemed positive for MMR‐related proteins, while cases in which the cytoplasm was stained were deemed positive for PTEN.