Corrigendum: Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

[This corrects the article DOI: 10.3389/fmolb.2023.1082915.].

Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

Background: Around 40% of ER+/HER2-breast carcinomas (BC) present mutations in the PIK3CA gene. Assessment of PIK3CA mutational status is required to identify patients eligible for treatment with PI3Kα inhibitors, with alpelisib currently the only approved tyrosine kinase inhibitor in this setting. U-PIK project aimed to conduct a ring trial to validate and implement the PIK3CA mutation testing in several Portuguese centers, decentralizing it and optimizing its quality at national level. Methods: Eight Tester centers selected two samples of patients with advanced ER+/HER2- BC and generated eight replicates of each (n = 16). PIK3CA mutational status was assessed in two rounds. Six centers used the cobas® PIK3CA mutation test, and two used PCR and Sanger sequencing. In parallel, two reference centers (IPATIMUP and the Portuguese Institute of Oncology [IPO]-Porto) performed PIK3CA mutation testing by NGS in the two rounds. The quality of molecular reports describing the results was also assessed. Testing results and molecular reports were received and analyzed by U-PIK coordinators: IPATIMUP, IPO-Porto, and IPO-Lisboa. Results: Overall, five centers achieved a concordance rate with NGS results (allele frequency [AF] ≥5%) of 100%, one of 94%, one of 93%, and one of 87.5%, considering the overall performance in the two testing rounds. NGS reassessment of discrepancies in the results of the methods used by the Tester centers and the reference centers identified one probable false positive and two mutations with low AF (1-3%, at the analytical sensitivity threshold), interpreted as subclonal variants with heterogeneous representation in the tissue sections processed by the respective centers. The analysis of molecular reports revealed the need to implement the use of appropriate sequence variant nomenclature with the identification of reference sequences (HGVS-nomenclature) and to state the tumor cell content in each sample. Conclusion: The concordance rates between the method used by each tester center and NGS validate the use of the PIK3CA mutational status test performed at these centers in clinical practice in patients with advanced ER+/HER2- BC.

BR-BCSC Signature: The Cancer Stem Cell Profile Enriched in Brain Metastases that Predicts a Worse Prognosis in Lymph Node-Positive Breast Cancer.

Brain metastases remain an unmet clinical need in breast oncology, being frequently found in HER2-overexpressing and triple-negative carcinomas. These tumors were reported to be highly cancer stem-like cell-enriched, suggesting that brain metastases probably arise by the seeding of cancer cells with stem features. Accordingly, we found that brain-tropic breast cancer cells show increased stem cell activity and tumorigenic capacity in the chick embryo choriallantoic membrane when compared to the parental cell line. These observations were supported by a significant increase in their stem cell frequency and by the enrichment for the breast cancer stem cell (BCSC) phenotype CD44+CD24-/low. Based on this data, the expression of BCSC markers (CD44, CD49f, P-cadherin, EpCAM, and ALDH1) was determined and found to be significantly enriched in breast cancer brain metastases when compared to primary tumors. Therefore, a brain (BR)-BCSC signature was defined (3-5 BCSC markers), which showed to be associated with decreased brain metastases-free and overall survival. Interestingly, this signature significantly predicted a worse prognosis in lymph node-positive patients, acting as an independent prognostic factor. Thus, an enrichment of a BCSC signature was found in brain metastases, which can be used as a new prognostic factor in clinically challenging breast cancer patients.

360 Health Analysis (H360) - A Proposal for an Integrated Vision of Breast Cancer in Portugal.

H360 aims to provide a comprehensive picture of breast cancer management in Portugal by retrieving real-world data from 10 Portuguese hospitals and deriving a snapshot from the medical interpretation of evidence-based data to patient perspective on the quality and effectiveness of medical care provided. This article reviews evidence on breast cancer clinical practice and quality of care and disease management in Portugal. A review of evidence on breast cancer clinical practice and quality of care over the last 10 years was performed in PubMed using the query "Organization and Administration"[Mesh] AND "breast cancer"[All Fields] NOT "Review" [ptyp]. National cancer initiatives relevant for quality of care and national and international guidelines and consensus were analyzed. Retrieved results showed that breast cancer incidence is still increasing, including in Portugal. Studies investigating disease outcomes seek to derive improvements to clinical practice and better financial resource allocation. Setting performance measures (KPIs) in institutions treating cancer is not a reality in Portugal yet, but has potential to leverage the quality of clinical performance. A multidisciplinary approach within one health structure is also desirable. More investment in clinical (including academic) research is key to optimize the quality of care. Implementation of clinical practice guidelines (largely based on ESMO guidelines in Portugal) is crucial to improve patient outcomes. Not less importantly, quality of life is a treatment goal on its own in breast cancer care. Breast cancer remains a health challenge and a multidimensional, 360-degree appraisal, beyond the exclusively clinical perspective, may provide new insights towards an optimal patient-centered approach.

Clinical and translational pharmacology of drugs for the prevention and treatment of bone metastases and cancer-induced bone loss.

Bone disease is a frequent event in cancer patients, both due to cancer spread to bone and to cancer therapies. Bone is the organ most frequently affected by metastatic disease when considering the two most frequent cancers in the Western world (breast and prostate cancers). Bone metastases can have a substantial detrimental effect on patients' quality of life, as well as significant morbidity due to complications collectively known as skeletal-related events (SREs), which include hypercalcaemia, pathological fractures, spinal cord compression, and need of radiotherapy or surgery to the bone. These have been successfully mitigated with the development of bone-targeted agents (BTAs; bisphosphonates and denosumab), focused on inhibiting osteoclast activity. The potential direct antitumour effect of bisphosphonates, as well as the impact of osteoclast inhibition with subsequent decrease in bone metabolism, have also propelled investigation on the role of BTAs in preventing cancer relapse in bone. In this review, the authors aimed to discuss the role of BTAs in the treatment and prevention of bone metastases, as well as their potential value in preventing cancer treatment-induced bone loss (CTIBL). The review will focus on breast and prostate cancers, with the aim of providing the most relevant clinical data emerging from bench to bedside translational research in the field of cancer-induced bone disease.

P-cadherin: a useful biomarker for axillary-based breast cancer decisions in the clinical practice.

Axillary lymph node metastases represent the most powerful breast cancer prognostic factor, dictating disease staging and clinical therapeutic decisions. Nonetheless, breast cancer patients with positive lymph nodes still exhibit a heterogeneous behavior regarding disease progression. Stem-like subpopulations of cancer cells show high migratory and metastatic capacity, thus we hypothesize that breast cancer stem cell markers evaluation in metastasized lymph nodes could provide a more accurate prediction of patient's prognosis. Therefore, the expression profile of P-cadherin, CD44, and CD49f, which have been already associated to stem cell properties in breast cancer, has been evaluated by immunohistochemistry in a series of 135 primary tumors and matched axillary lymph node metastases from 135 breast cancer patients. Taking in consideration the expression of the stem cell markers only in axillary nodes, P-cadherin was the only biomarker significantly associated with poor disease-free and overall patient's survival. Moreover, although a concordant expression between primary tumors and matched lymph nodes has been found in the majority of the cases, a small but significant percentage displayed divergent expression (18.2-26.2%). Remarkably, although CD44 and CD49f changes between primary tumors and lymph node metastasis did not impact survival, the cases that were positive for P-cadherin in lymph node metastases being negative in the primary tumor, presented the worst disease-free and overall survival of the whole series. Accordingly, negative cases for this marker in the lymph nodes with positive expression in the matched breast carcinoma demonstrated a better prognosis, which overlapped with tumors that were negative in both sites. P-cadherin and CD49f gain of expression was mainly found in triple-negative carcinomas. Our results indicate for the first time that the evaluation of P-cadherin expression in lymph node metastases is an important predictor of disease outcome, being a putative valuable marker for axillary-based breast cancer decisions in the clinical practice.

Metronomic chemotherapy for advanced breast cancer patients.

Metronomic chemotherapy refers to the minimum biologically effective dose of a chemotherapy agent given as a continuous dosing regimen with no prolonged drug-free breaks that leads to antitumor activity. This schedule seems to have not only a direct cytotoxicity on cancer cells but also an effect on the tumor microenvironment by inhibiting tumor angiogenesis and modulating immune response. Metronomic chemotherapy was widely investigated in patients with breast cancer. The results of these studies showed that this strategy is not only effective but has a low toxicity profile too, proposing as a promising strategy for breast cancer patients. In this review we summarize the results of Phase II and III studies evaluating metronomic therapy in metastatic breast cancer.

P-cadherin signals through the laminin receptor α6ß4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells.

P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor α6ß4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the α6 integrin subunit expression and directly interacts with the ß4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, α6ß4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor α6ß4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule.

Cancer stem cell markers in breast neoplasias: their relevance and distribution in distinct molecular subtypes.

The identification and characterization of cancer stem cells might lead to more effective control of neoplastic disease, by directing therapies to the most aggressive cells. For that reason, the identification of cancer stem cells (CSCs) in breast tumours is one of the priorities in breast cancer research, which has resulted in many studies attempting to identify their presence based on the expression of specific molecular markers. In this review, we describe the main molecular markers that have been identified as being able to recognise CSCs in breast carcinomas, the major molecular pathways that regulate CSCs and their association with the different molecular subtypes.

P-cadherin is coexpressed with CD44 and CD49f and mediates stem cell properties in basal-like breast cancer.

Although the luminal progenitor cell of the normal mammary gland hierarchy has been proposed as the cell-of-origin for basal-like breast cancers, finding the cancer stem cell (CSC) phenotype for this malignancy has proven a difficult task, mostly due to the lack of specific markers. Recently, basal-like sporadic and familial cases of breast cancer have been linked to BRCA1 gene inactivation, which enables the upregulation of the target-repressed CDH3/P-cadherin gene, an important biomarker of basal-like breast carcinomas. Previously, we demonstrated that P-cadherin overexpression can mediate aggressive behavior in these tumors. Thus, our aim was to test whether P-cadherin mediates stem cell properties in basal-like breast carcinomas. Using a series of breast cancer cell lines and primary tumors, we showed that P-cadherin was directly associated with the expression of the breast stem markers CD44, CD49f, and aldehyde dehydrogenase 1 in the basal subtype. Moreover, cell population enriched for P-cadherin expression comprised increased in vitro mammosphere-forming efficiency and capacity to grow colonies in three-dimensional cultures as well as greater tumorigenicity. Importantly, an association was found with stem-/progenitor-like phenotypes of the breast, including the luminal progenitor population, CD49f(+) CD24(+). Additionally, P-cadherin expression conferred resistance to x-ray-induced cell death, sustaining a role for this molecule in another stem cell property. In summary, we demonstrated, for the first time, that P-cadherin mediates stem cell properties, which could be explored in order to better define the CSC phenotype of basal-like breast tumors and the cell-of-origin of this malignancy.