A CD44?/CD24+ phenotype is a poor prognostic marker in early invasive breast cancer

A CD44(-)/CD24(+) phenotype is a poor prognostic marker in early invasive breast cancer. Breast cancer cells with high CD44 and low or absent CD24 (i.e. CD44(+)CD24(-)/low phenotype) are reported to have stem cell features. However, the clinical impact of CD24 and CD44 expression in tumours remains unclear. To explore the immunohistochemical expression of CD44 and CD24 (individually and combined) and their clinical value as prognostic and predictive markers. Immunohistochemical expression of CD24 and CD44 was studied in a large series of early primary invasive breast cancer tumours (n = 1036) prepared as a tissue microarray. Associations between the expression of each marker individually and in combination and clinico-pathological, molecular variables and patients' outcome were investigated. CD24 cytoplasmic expression was significantly associated with poor prognostic variables including high tumour grade, ER-, PR-, HER2(+), p53+ and triple negative (TN) phenotype; P < 0.05. However, CD24 expression was not significantly associated with patients' outcome. Conversely, CD44 expression was associated with favourable prognostic criteria including lower Nottingham prognostic index, ER+, HER2- and luminal phenotype; P < 0.05. Moreover, CD44 expression was found to be an independent predictor of good prognosis. In combination, the CD44(+)/CD24(-) phenotype was associated with the most favourable outcome (84 and 80% 10 year breast cancer survival [BCSS] and metastasis free survival [MFS], respectively). Contrasting this, the CD44(-)/CD24(+) phenotype was associated with the most dismal outcome (62 and 60% 10 years BCSS and MFS, respectively). CD24 and CD44 expression can individually yield prognostic data in breast cancer, but importantly, when both markers are considered; the CD44(+)/CD24(-) phenotype had the best prognosis, while the CD44(-)/CD24(+) phenotype had the worst prognosis. This shows that the relationship between basic cell biology and clinical behaviour is not always straightforward and warrants further investigations of the true clinical impact of breast cancer stem cells.     

Use of standard markers and incorporation of molecular markers into breast cancer therapy: Consensus recommendations from an International Expert Panel

Breast cancer is a heterogeneous disease of different subtypes on the molecular, histopathological, and clinical level. Genomic profiling techniques have led to several prognostic and predictive gene signatures of breast cancer that may further refine outcome prediction, especially in clinically equivocal situations. In particular, the predictive value of today's most important therapeutic targets, ER and HER2, are strongly influenced by the proliferative status of the tumor. Genomic assays are generally performed in a centralized manner, whereas routine pathological evaluation is mostly done on a decentralized basis, making the comparison of these methods difficult. Thus, there remains considerable uncertainty about the use of the new molecular markers in routine clinical decision making and their role in patient selection or stratification for future clinical trials. To address this concern, a group of representatives from breast cancer research groups in the areas of breast pathology, genomic profiling, and clinical trials critically reviewed all available data. Consensus recommendations are made on the practical use of molecular markers in breast cancer management and their incorporation into future clinical trials.     

Triple-negative breast cancers: an updated review on treatment options

Morphologic features of tumour cells have long been validated for the clinical classification of breast cancers and are regularly used as a "gold standard" to ascertain prognostic outcome in patients. Identification of molecular markers such as expression of the receptors for estrogen (er) and progesterone (pgr) and the human epidermal growth factor receptor 2 (her2) has played an important role in determining targets for the development of efficacious drugs for treatment and has also offered additional predictive value for the therapeutic assessment of patients with breast cancer. More recent technical advancements in identifying several cancer-related genes have provided further opportunities to identify specific subtypes of breast cancer. Among the subtypes, tumours with triple-negative cells are identified using specific staining procedures for basal markers such as cytokeratin 5 and 6 and the absence of er, pgr, and her2 expression. Patients with triple-negative breast cancers therefore have the disadvantage of not benefiting from currently available receptor-targeted systemic therapy. Optimal conditions for the therapeutic assessment of women with triple-negative breast tumours and for the management of their disease have yet to be validated in prospective investigations. The present review discusses the differences between triple-negative breast tumours and basal-like breast tumours and also the role of mutations in the BRCA genes. Attention is also paid to treatment options available to patients with triple-negative breast tumours.     

Emerging Biomarkers of the Future: Changing Clinical Practice for 2020

Progress in biomarker development has greatly enhanced our ability to categorize breast cancer into several clinical subtypes and to deliver better personalized therapies. Technological advances in gene expression profiling, signaling pathways, proliferation markers and tumor monitoring through detection of circulating tumor cells and free DNA, and measurements of genomic instability and germline mutations are being vigorously pursued in breast cancer research. Their application in routine clinical practice is increasing and helping further development of precision medicine. Ongoing challenges include assessing the utility and feasibility of these tests, interpreting the large amounts of genomic data that are being generated, translating the information to clinical practice, and constructing clinical trials on molecularly driven approaches. In this article, we will review current and emerging promising biomarkers and their roles in the management of patients with breast cancer.     

Breast Cancer Classification to Select Patients for Cancer Treatment

Gene expression arrays have shown that breast cancer is comprised of at least four different molecular diseases. These are 1) basal-like breast cancer, 2) human epidermal growth factor receptor 2 (Her2)-positive/estrogen receptor (ER)-negative breast cancer; 3) luminal A breast cancer; and 4) luminal breast B cancer. Basal-like cancer is characterized by ER-negative, progesterone receptor–negative, and Her2-negative expressions. Luminal breast cancer is characterized by ER-positive expression, with the luminal B subtype exhibiting a higher expression of proliferation genes. Several studies have shown that luminal A breast cancer exhibits a better outcome compared with other classes. Also, recent studies have shown that the same subclass is less chemosensitive. These data suggest that some selected luminal A breast cancer cases could be spared adjuvant chemotherapy given their better outcome and lesser chemosensitivity compared with other subtypes of breast cancer. Although other subtypes are more chemosensitive, there is debate about whether some drugs could be active in specific subtypes. Anthracyclines have been reported to be more effective in Her2-overexpressing breast cancer, and cisplatin is currently under investigation in basal-like breast cancer. Finally, some data suggest that luminal B breast cancer exhibits higher sensitivity to aromatase inhibitors. Overall, molecular classes exhibit different sensitivity to conventional drugs. To what extent these data obtained in retrospective biomarker studies could be implemented in daily practice is a matter of debate given the heterogeneity of findings in different studies.     

Estrogen and progesterone receptor isoforms: clinical significance in breast cancer

The identification and exploitation of biomarkers that may predict response to anti-cancer treatments has the capacity to revolutionize the way that patients with cancer are treated. In breast cancer, the estrogen receptor (ER) and the progesterone receptor (PgR) are known to have a significant predictive value in determining sensitivity to endocrine therapies. Tumor expression of ER or PgR is known to affect clinical outcome and this information is often used to determine a patient's optimal treatment regimen. However, the measurement of ER and PgR alone is more complex than originally thought and the impact of the recently identified isoforms of ER (ERα and ERβ) and PgR (PgRA and PgRB), as well as several variant and mutant forms, upon the choice of treatment remains unclear. Therefore, ER and PgR expression alone are unlikely to determine a patient's optimal treatment regimen, particularly when the amount of ‘cross-talk’ between different pathways, such as the epidermal growth factor receptor pathway, is considered. In order to account for the complex cell-signaling environment that occurs in breast cancer, multifactorial techniques are needed to analyze tumor biomarker expression. The recent advances in genomic- or proteomic-based approaches has enabled molecular portraits of breast cancers to be painted, allowing biomarkers of response and prognosis to be identified and characterized more accurately than before. In the future, patients could be treated according to the molecular portrait of their tumor biomarker expression, maximizing the therapeutic benefit that each patient receives. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neoadjuvant treatment for advanced esophageal cancer: response assessment before surgery and how to predict response to chemoradiation before starting treatment

Patients with advanced esophageal cancer (T3-4, N) have a poor prognosis. Chemoradiation or chemotherapy before esophagectomy with adequate lymphadenectomy is the standard treatment for patients with resectable advanced esophageal carcinoma. However, only patients with major histopathologic response (regression to less than 10% of the primary tumor) after preoperative treatment will have a prognostic benefit of preoperative chemoradiation. Using current therapy regimens about 40% to 50% of the patients show major histopathological response. The remaining cohort does not benefit from this neoadjuvant approach but might benefit from earlier surgical resection. Therefore, it is an aim to develop tools for response prediction before starting the treatment and for early response assessment identifying responders. The current review discusses the different imaging techniques and the most recent studies about molecular markers for early response prediction. The results show that [¹⁸F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) has a good sensitivity but the specificity is not robust enough for routine clinical use. Newer positron emission tomography detector technology, the combination of FDG-PET with computed tomography, additional evaluation criteria and standardization of evaluation may improve the predictive value. There exist a great number of retrospective studies using molecular markers for prediction of response. Until now the clinical use is missing. But the results of first prospective studies are promising. A future perspective may be the combination of imaging technics and special molecular markers for individualized therapy. Another aspect is the response assessment after finishing neoadjuvant treatment protocol. The different clinical methods are discussed. The results show that until now no non-invasive method is valid enough to assess complete histopathological response.     

The scientific value of preoperative studies and how they can be used

Preoperative hormonal therapy appears to be an effective option for the treatment of breast cancer and this treatment approach enables breast conservation surgery to be performed instead of mastectomy in patients with large, operable tumors. Short-term preoperative trials are valuable for research of established agents where differential efficacy in biologically distinct subgroups is to be investigated. They can be used to generate hypotheses that can be further examined in suitably powered clinical trials. Longer-term ‘thera-peutic trials’, in which surgery is delayed over a period of months, not only allow for a reduction in tumor size with treatment, but also provide an opportunity to perform a detailed analysis of biomarker expression. Recent advances in hormonal preoperative studies and the increasing importance of prognostic and predictive factors in the management of patients with breast cancer are discussed in this review. Indeed, several clinical studies have been performed in the preoperative setting and it is now apparent that the aromatase inhibitors, such as letrozole (‘Femara’) and anastrozole (‘Arimidex’), are valid new treatment options at this stage of treatment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular and genetic markers in the local-regional management of breast cancer

The clinical application of molecular markers in the diagnosis, staging, and management of breast cancer continues to expand. Although the use of molecular markers in local-regional disease does not approach the level of their application in the systemic management of breast cancer, a growing body of rature supports the potential for molecular and genetic factors in clinical decision making regarding the local-regional management of breast cancer. As with conventional clinical and histopathologic factors, data regarding molecular and genetic factors as they relate to local-regional relapse may be conflicting and are subject to the usual limitations of predominantly retrospective studies. There are, however, some consistent data suggesting associations between local-regional control of disease and several molecular markers, including hormone receptor status, HER2/neu, p53, proliferative markers, and others. Interpretation of these data and how to use this information in clinical practice remains challenging. The available rature regarding the use of genetic and molecular markers in the local-regional management of breast cancer is summarized in this review.     

Clinical challenges in the molecular characterization of circulating tumour cells in breast cancer

Blood testing for circulating tumour cells (CTC) has emerged as one of the hottest fields in cancer research. CTC detection and enumeration can serve as a ‘liquid biopsy'' and an early marker of response to systemic therapy, whereas their molecular characterisation has a strong potential to be translated to individualised targeted treatments and spare breast cancer (BC) patients unnecessary and ineffective therapies. Different analytical systems for CTC detection and isolation have been developed and new areas of research are directed towards developing novel assays for CTC molecular characterisation. Molecular characterisation of single CTC holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. The application of extremely powerful next-generation sequencing technologies in the area of CTC molecular characterisation in combination with reliable single CTC isolation opens new frontiers for the management of patients in the near future. This review is mainly focused on the clinical potential of the molecular characterisation of CTC in BC.     

乳腺癌血清肿瘤标志物和循环肿瘤细胞检测的研究进展

 随着分子生物学的发展,乳腺癌的治疗正逐步进入分子分型治疗的时代,为了给患者提供更加有效的个体化治疗,研究者们不断努力寻找乳腺癌新的治疗靶点、疾病监控指标以及疗效预测和预后评估的指标,日益深入的研究显示血清肿瘤标志物和循环肿瘤细胞的检测在乳腺癌治疗中有重要价值。现就近年乳腺癌血清肿瘤标志物和循环肿瘤细胞检测的相关临床研究作一综述。     

Molecular markers in the diagnosis and staging of breast cancer

Considerable advances have been made in understanding the molecular events that accompany the development of breast cancer. Although our knowledge of these genetic alterations has greatly outpaced clinical applications, many new advances are beginning to have an impact on the diagnosis and staging of breast cancer. Clinical evaluation of estrogen and progesterone receptors and HER2/neu status has become routine. The increasing use of these and other molecular markers promises to help refine diagnoses, define disease subsets, and provide more accurate information about the probable biologic outcome of a given tumor. Studies of molecular markers are also likely to lead to the identification and development of new therapeutic targets. I review the molecular markers currently used in the clinical diagnosis and staging of breast cancer, and discuss other potentially useful markers and assays.     

Molecular classification and molecular forecasting of breast cancer: ready for clinical application?

Profiling breast cancer with expression arrays has become common, and it has been suggested that the results from early studies will lead to understanding of the molecular differences between clinical cases and allow individualization of care. We critically review two main applications of expression profiling; studies unraveling novel breast cancer classifications and those that aim to identify novel markers for prediction of clinical outcome. Breast cancer may now be subclassified into luminal, basal, and HER2 subtypes with distinct differences in prognosis and response to therapy. However, profiling studies to identify predictive markers have suffered from methodologic problems that prevent general application of their results. Future work will need to reanalyze existing microarray data sets to identify more representative sets of candidate genes for use as prognostic signatures and will need to take into account the new knowledge of molecular subtypes of breast cancer when assessing predictive effects.     

Role of biologic markers in patient selection and application to disease prevention

Aromatase inhibitors (AIs) are now under investigation for the treatment of early stage breast cancer and for disease prevention as alternatives to standard treatment with tamoxifen. Currently identified genetic risk factors of breast cancer include BRCA-1/BRCA-2 mutations, ATM mutations, and history of high estrogen levels, as evidenced by plasma analyses and/or dense bones. To date, estrogen receptor (ER) and progesterone receptor (PgR) status has predictive value for determining response to therapy in patients with hormone receptor-positive breast cancer (ER+ and/or PgR+ tumors). Recent studies have shown AIs to be safer and more effective than tamoxifen in postmenopausal women with advanced disease. Some data suggest that letrozole may be a more effective treatment than tamoxifen for patients with ER+ and/or PgR+ early breast cancers expressing ErbB-1 and/or ErbB-2. Changes in cell proliferation markers (e.g., S-phase fraction and Ki67 antigen), plasma lipid levels, and the bone resorption marker C-terminal peptide are biomarkers that have been evaluated for preventive and prognostic value in breast cancer patients and normal volunteers. Results from biomarker screens can be used to define inclusion criteria for clinical trials and eventually to individualize treatment. Gene expression profiling (microarray analysis), i.e., genomic and proteomic studies, will probably advance the discovery of new biomarkers for breast cancer prevention and treatment.     

Clinical Utility of Serum Tumor Markers and Circulating Tumor Cell Assays in the Treatment of Breast Cancer

Though serum tumor markers and circulating tumor cells (CTCs) have been available to the oncologist for many years, their place in the management of breast cancer remains unclear. Due to issues with sensitivity and specificity, tumor markers are unreliable for the detection of metastases in early stage breast cancer. For patients with metastatic breast cancer without measurable disease (e.g., bone-only disease, pleural effusions, or ascites), it is reasonable to obtain baseline values of serum tumor markers and attempt to correlate them with the first set of scans. In patients with elevated markers at baseline whose tumor marker levels decrease in correlation with an improvement in clinical symptoms, it may be reasonable to use them in conjunction with imaging to help determine whether there is progression of disease. CTCs have been found to hold strong prognostic value for breast cancer in both the early stage and metastatic settings. No large prospective studies to date, however, have shown any predictive value for CTCs and their clinical utility is therefore limited. Whether changing treatment in response to an increase in CTCs without radiologic progression results in improvements in quality of life or survival remains to be seen. CTCs hold great promise in the management of breast cancer and future studies will help delineate their role more appropriately.     

Alterations of biomarker profiles after neoadjuvant chemotherapy in breast cancer: tumor heterogeneity should be taken into consideration

Tumor biomarkers including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely tested in breast cancer patients and their status guides clinical management and predicts prognosis. A few retrospective studies have suggested that neoadjuvant chemotherapy (NAC) in breast cancer may change the status of biomarker expression, which in turn will affect further management of these patients. In this study we take advantage of a relatively large cohort and aim to study the effect of NAC on biomarker expression and explore the impact of tumor size and lymph node involvement on biomarker status changes. We collected 107 patients with invasive breast cancer who received at least three cycles of NAC. We retrospectively performed and scored the immunohistochemistry (IHC) of ER, PR, HER2 and Ki-67 using both the diagnostic core biopsies before NAC and excisional specimens following NAC. HER2 gene status was assessed by fluorescence in situ hybridization for cases with IHC result of 2+. We demonstrated that there was a significant decrease in expression of PR (P = 0.013) and Ki-67 (P = 0.000) in post-NAC specimens compared to pre-NAC core biopsies. In addition, cases with large tumor size (≥2cm) and cases with lymph node metastasis were more frequently to have biomarker changes. Finally we studied cases with HER2 status changes after NAC treatments in detail and emphasized the nature of tumor heterogeneity.     

Targeting breast stem cells with the cancer preventive compounds curcumin and piperine

The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin, and piperine are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and Wnt signaling. Mammosphere formation assays were performed after curcumin, piperine, and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation, serial passaging, and percent of ALDH+ cells by 50% at 5 μM and completely at 10 μM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 μM and completely at 10 μM. Curcumin and piperine separately, and in combination, inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials.     

Classification of breast cancer using genetic algorithms and tissue microarrays.

PURPOSE: A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome. EXPERIMENTAL DESIGN: We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section. RESULTS: The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149). CONCLUSIONS: With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.     

Prognostic molecular markers in early breast cancer

A multitude of molecules involved in breast cancer biology have been studied as potential prognostic markers. In the present review we discuss the role of established molecular markers, as well as potential applications of emerging new technologies. Those molecules used routinely to make treatment decisions in patients with early-stage breast cancer include markers of proliferation (e.g. Ki-67), hormone receptors, and the human epidermal growth factor receptor 2. Tumor markers shown to have prognostic value but not used routinely include cyclin D₁ and cyclin E, urokinase-like plasminogen activator/plasminogen activator inhibitor, and cathepsin D. The level of evidence for other molecular markers is lower, in part because most studies were retrospective and not adequately powered, making their findings unsuitable for choosing treatments for individual patients. Gene microarrays have been successfuly used to classify breast cancers into subtypes with specific gene expression profiles and to evaluate prognosis. RT-PCR has also been used to evaluate expression of multiple genes in archival tissue. Proteomics technologies are in development.     

Immune infiltrates in the breast cancer microenvironment: detection,characterization and clinical implication

Although unlike melanoma, breast cancer is not generally viewed as a highly immunogenic cancer, recent studies have described a rich tumor immune microenvironment in a subset of breast cancers. These immune infiltrates, comprised cells from the innate and adaptive immune response, can be detected and characterized in biopsy specimens and have prognostic value. Tumor-infiltrating lymphocytes (TILs) represent the majority of mononuclear immune infiltrates in the breast tumor microenvironment and can be easily identified in formalin-fixed paraffin-embedded tissues after standard hematoxylin and eosin staining. High levels of TILs are most common in HER2+ and basal-like subtypes where they are associated with good prognosis and with response to certain therapies such as the anti-HER2 antibody trastuzumab. International collaborative efforts are underway to standardize the assessment of TILs so as to facilitate their implementation as a breast cancer biomarker. Using immunohistochemistry to further characterize TILs, recent reports describe the presence of important lymphocyte populations including CD8+ cytotoxic, FOXP3+ regulatory, and CD4+ helper and follicular T cells which have overlapping associations with prognosis and response to therapies. Moreover, recently identified immune checkpoint markers (PD-1, PD-L1) are present in some breast cancers, implying some cases might be especially amenable to immune checkpoint inhibitor treatment strategies which are being evaluated in a number of active clinical trials.