Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway either through loss of PTEN or mutation of the catalytic subunit α of PI3K (PIK3CA) occurs frequently in human cancer. We identified PIK3CA mutations in 26% of 342 human breast tumor samples and cell lines at about equal frequency in tumor stages I to IV. To investigate the relationship between PTEN and PIK3CA, we generated a cohort of tumors that had lost PTEN expression and compared it with a matched control set that had retained PTEN. A highly significant association between PIK3CA mutations and retention of PTEN protein expression was observed. In addition, PIK3CA mutations were associated with expression of estrogen and progesterone receptors (ER/PR), lymph node metastasis, and ERBB2 overexpression. The fact that PIK3CA mutations and PTEN loss are nearly mutually exclusive implies that deregulated phosphatidylinositol-3,4,5-triphosphate (PIP3) is critical for tumorigenesis in a significant fraction of breast cancers and that loss of PIP3 homeostasis by abrogation of either PIK3CA or PTEN relieves selective pressure for targeting of the other gene. The correlation of PIK3CA mutation to ER/PR-positive tumors and PTEN loss to ER/PR-negative tumors argues for disparate branches of tumor evolution. Furthermore, the association between ERBB2 overexpression and PIK3CA mutation implies that more than one input activating the PI3K/AKT pathway may be required to overcome intact PTEN. Thus, mutation of PIK3CA is frequent, occurs early in carcinoma development, and has prognostic and therapeutic implications.

Introduction: Human breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44⁺/CD24^- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes. Methods: Double-staining immunohistochemistry was used to quantify CD44 and CD24 expression in 240 human breast tumors for which information on other tumor markers and clinical characteristics was available. Gene expression data were also accessible for a cohort of the material. Results: A considerable heterogeneity in CD44 and CD24 expression was seen both between and within tumors. A complete lack of both proteins was evident in 35% of the tumors, while 13% contained cells of more than one of the CD44⁺/CD24^-, CD44^-/CD24⁺ and CD44⁺/CD24⁺ phenotypes. CD44⁺/CD24^- cells were detected in 31% of the tumors, ranging in proportion from only a few to close to 100% of tumor cells. The CD44⁺/CD24^- phenotype was most common in the basal-like subgroup – characterized as negative for the estrogen and progesterone receptors as well as for HER2, and as positive for cytokeratin 5/14 and/or epidermal growth factor receptor, and particularly common in BRCA1 hereditary tumors, of which 94% contained CD44⁺/CD24^- cells. The CD44⁺/CD24^- phenotype was surprisingly scarce in HER2+ tumors, which had a predominantly CD24⁺ status. A CD44⁺/CD24^- gene expression signature was generated, which included CD44 and α₆-integrin (CD49f) among the top-ranked overexpressed genes. Conclusion: We demonstrate an association between basal-like and particularly BRCA1 hereditary breast cancer and the presence of CD44⁺/CD24^- cells. Not all basal-like tumors and very few HER2+ tumors, however, contain CD44⁺/CD24^- cells, emphasizing that a putative tumorigenic ability may not be confined to cells of this phenotype and that other breast cancer stem cell markers remain to be identified.

Pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2 . This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1 , was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo . Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

Metastatic breast cancer is usually diagnosed after becoming symptomatic, at which point it is rarely curable. Cell‐free circulating tumor DNA (ctDNA) contains tumor‐specific chromosomal rearrangements that may be interrogated in blood plasma. We evaluated serial monitoring of ctDNA for earlier detection of metastasis in a retrospective study of 20 patients diagnosed with primary breast cancer and long follow‐up. Using an approach combining low‐coverage whole‐genome sequencing of primary tumors and quantification of tumor‐specific rearrangements in plasma by droplet digital PCR, we identify for the first time that ctDNA monitoring is highly accurate for postsurgical discrimination between patients with (93%) and without (100%) eventual clinically detected recurrence. ctDNA‐based detection preceded clinical detection of metastasis in 86% of patients with an average lead time of 11 months (range 0–37 months), whereas patients with long‐term disease‐free survival had undetectable ctDNA postoperatively. ctDNA quantity was predictive of poor survival. These findings establish the rationale for larger validation studies in early breast cancer to evaluate ctDNA as a monitoring tool for early metastasis detection, therapy modification, and to aid in avoidance of overtreatment.

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis^1,2,3. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype^3,4,5; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.