Immunohistochemistry for PTEN testing in HR +/HER2˗ metastatic breast cancer

The PTEN tumor suppressor gene plays a Major role in regulating the phosphatidylinositol 3-kinase (PI3K) pathway and its oncogenic signaling [1]. In breast cancer, PTEN inactivation, mainly through gene deletions, occurs in 5% of hormone receptor (HR) +/HER2- metastatic breast cancer (MBC) and leads to tumorigenesis and tumor progression [2]. At present, three therapeutic options targeting the PI3K pathway are approved by both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of these patients [3]. Among these are Alpelisib and Inavolisib, which specifically inhibit the alpha isoform of PIK3CA, as well as Capivasertib, a first-in-class pan-AKT inhibitor [4‐6]. Notably, Capivasertib has three potential biomarkers for patients’ selection: PIK3CA/AKT1 activating mutations and PTEN loss of function [7]. In the CAPItello-291 study (NCT04305496), all these alterations were determined centrally by means of next-generation sequencing (NGS) with the use of the FoundationOne CDx assay on formalin-fixed paraffin-embedded (FFPE) tumor samples [6, 8].

In most pathology laboratories worldwide, immunohistochemistry (IHC) is widely used not only in diagnostic and predictive pathology but also as a surrogate for molecular classifications, due to its accessibility and cost-effectiveness [9‐12]. For example, assessing PTEN protein loss through ancillary IHC analyses is routinely performed for gynecologic tumors [13, 14]. In an exploratory analysis presented at the San Antonio Breast Cancer Symposium 2024, the CAPItello-291 investigators used the Ventana PTEN SP218 IHC assay for PTEN testing [15]. Among samples with both NGS and IHC data, all cases with homozygous deletions or large PTEN rearrangements detected by NGS were classified as PTEN deficient by IHC (defined as < 10% staining in tumor cells); however, the reverse relationship was not consistently evident. Of note, PTEN-deficient tumors by IHC showed a promising progression-free survival (PFS) benefit with capivasertib plus fulvestrant compared to placebo plus fulvestrant (median PFS of 9.3 months versus 3.7 months; hazard ratio: 0.52.

While NGS should be considered the preferred testing method for breast cancer molecular testing [16, 17], IHC can still serve as a complementary approach, particularly in settings where NGS is not available, cost-prohibitive, or impractical due to limited tissue availability. Additionally, IHC offers a rapid and widely accessible method for assessing protein expression, which may provide clinically relevant insights, especially when timely treatment decisions are required. However, standardized protocols are essential to ensure accurate, reproducible, and clinically meaningful PTEN deficiency analysis by IHC in HR +/HER2 − MBC, minimizing variability and enhancing diagnostic accuracy.

PTEN is encoded by a single gene but multiple protein isoforms arise through alternative mRNA splicing or variations in translation initiation [18]. The most prevalent one, consisting of 403 amino acids (PTEN 1–403), is the primary form referred to in the literature on human cancers [19‐21]. Numerous anti-PTEN monoclonal antibodies are available and have been variably used in breast cancer studies using IHC [22‐42]. In addition to the variability in commercial PTEN assays, the broad spectrum of PTEN IHC staining patterns observed in breast cancer may pose diagnostic challenges [43]. Another potential issue is represented by the handling and processing of tissue samples to obtain reliable and reproducible IHC results [44, 45]. Pre-analytical variables—such as cold ischemic time, fixation duration, type of fixative used, and storage conditions—can dramatically affect PTEN antigen preservation [46‐49]. Moreover, technical aspects of the staining procedure itself—including antigen retrieval methods, the selection of antibody clones, incubation times, temperature, and the detection system—play key roles in determining the intensity and quality of the staining [49‐51]. In the post-analytical phase, interobserver variability might further complicate the picture [52‐54]. These challenges underscore the need for rigorous quality control measures and the establishment of well-defined, standardized guidelines for PTEN IHC assessment specifically tailored to breast cancer [20, 55]. The implementation of strict standard operating procedures (SOPs) is pivotal in ensuring consistency and accuracy at every stage—from specimen collection and processing to staining and reporting [56‐61]. Participation in external quality assessment (EQA) programs, such as JCI, UK NEQAS, and NordiQC, is highly recommended [62‐67]. These programs provide benchmarking against standardized controls and help identify inconsistencies in staining and interpretation.

Standardized PTEN IHC testing plays a significant role in evaluating PI3K pathway alterations in HR +/HER2 − MBC, especially in laboratories without access to NGS, when archival tissue is inadequate for sequencing, or in case of pre-analytical failures, as observed in approximately 10% of samples in the CAPItello-291 trial. Thus, IHC is a feasible choice for routine clinical practice, whereas NGS is preferable.