High FOXA1 protein expression might predict late recurrence in patients with estrogen-positive and HER2-negative breast cancer.

BACKGROUND: Multi-gene expression assays have been developed with the aim of predicting late recurrence in patients with estrogen receptor (ER)-positive breast cancer. However, establishment of alternative markers based on immunohistochemistry is also important for achieving practical use. Based on our previous study, forkhead box A1 (FOXA1) protein was tested as a potentially useful predictive marker for late recurrence. METHODS: 117 patients with ER-positive HER2-negative invasive breast cancer who developed distant metastasis following curative surgery were retrospectively investigated. We also evaluated responsiveness to endocrine therapy according to FOXA1 expression. Furthermore, publicly available mRNA microarray data were analyzed to examine patterns of metastasis according to FOXA1 mRNA expression, employing the Kaplan-Meier plotter. RESULTS: High expression of FOXA1 was an independent factor predicting long disease-free survival (DFS), along with small tumor size (p = 0.010 and 0.016, respectively). Discrimination of DFS was improved by combining these two factors, i.e., patients with FOXA1-high small tumors had the longest DFS while those with FOXA1-low large tumors had the shortest DFS. Moreover, we revealed that risk of distant metastasis started to increase after the completion of adjuvant endocrine therapy in patients with FOXA1-high tumors. CONCLUSION: Among patients who developed distant metastasis, those with FOXA1-high tumors had significantly longer DFS. We believe our data to raise the possibility of FOXA1 being a useful predictive marker for late recurrence and to provide new insights into the biology of FOXA1-high breast cancers.

Neutrophil-to-lymphocyte ratio and histological type might predict clinical responses to eriburin-based treatment in patients with metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is generally considered to be incurable. Although many options are available for treating MBC, physicians often encounter difficulties in choosing the most appropriate treatment because the MBCs of individual patients respond differently even to the same treatments. Thus, predictive markers for therapeutic efficacy are urgently needed. Neutrophil- and platelet-to-lymphocyte ratios (NLR and PLR, respectively), have been studied and established as prognostic markers for breast cancer patients but whether either or both of these markers are predictive of treatment responses is still unclear. Herein, we investigated predictive markers for eribulin-based treatment responsiveness in patients with MBC, by examining clinicopathological features, including several markers of immunocompetent cells in peripheral blood. METHODS: Clinicopathological features of the 104 patients with metastatic/Stage IV breast cancer given eribulin-based regimens were investigated in relation to clinical responses to eribulin-based treatments and progression-free-survival (PFS). RESULTS: Special histological types and high NLR at baseline were independently related to poor clinical responses to the treatments (p = 0.023 and 0.039, respectively). The Cox hazard model revealed that patients with oestrogen receptor (ER)-negative tumours and high NLR, monocyte-to-lymphocyte ratio (MLR) and PLR showed significantly shorter PFS (p = 0.021, 0.005, 0.008 and 0.030, respectively). On multivariate analysis, only ER status and NLR remained independent factors related to PFS (p = 0.011 and 0.003, respectively). CONCLUSIONS: Our data revealed that special histological types and high NLR might be factors related to low responsiveness to eribulin-based regimens in patients with MBC.

Impact of circulating tumour cells on survival of eribulin-treated patients with metastatic breast cancer.

Several clinical studies have examined circulating tumour cells (CTCs). However, the application of CTCs as a predictive/prognostic marker for breast cancer patients has yet to be established, particularly the selection of suitable markers for detecting CTCs. We recently investigated CTCs, including mesenchymal status, from metastatic breast cancer patients who had received eribulin-based treatment. We found that assessment of both mesenchymal and epithelial CTCs might be important for predicting eribulin responsiveness. In the current study, we followed up the outcomes of these patients after eribulin treatment and investigated the possibility of CTC analysis results serving as prognostic markers for this patient population. Twenty-one patients were enrolled and peripheral blood samples were collected before eribulin-based treatments. CTCs were then examined using a Microfluidic Chip device. CTCs positive for vimentin and pan-cytokeratin were defined as mesenchymal and epithelial CTCs, respectively. Overall survival (OS) was assessed in relation to the number of CTCs and clinicopathological factors. During the observation period, 13 patients (62%) died due to breast cancer and the median OS was 18 months. Patients with high-grade tumours and a high total number of CTCs showed significantly shorter OS than those with low-grade tumours and smaller CTC burdens (p = 0.026 and 0.037, respectively). Patients who received eribulin as the first chemotherapy for metastatic disease showed longer OS (p = 0.006). Our data suggest that determining numbers of both mesenchymal and epithelial CTCs might predict survival for patients receiving eribulin.

Analysis of circulating tumour cell and the epithelial mesenchymal transition (EMT) status during eribulin-based treatment in 22 patients with metastatic breast cancer: a pilot study.

BACKGROUND: Liquid biopsy approaches, such as measuring circulating tumour cells (CTCs), have recently been introduced in several clinical studies. However, the development of CTCs as a predictive marker for treatment effects on breast cancer remains an enormous task. We investigated CTCs, including epithelial mesenchymal transition (EMT) status, from metastatic breast cancer patients who had received eribulin-based treatment, which reportedly suppresses EMT as a means of tumour suppression. Our aim was to test the possibility of this method serving as a tool predicting eribulin efficacy. METHODS: Twenty-two patients were enrolled and peripheral blood samples were collected before eribulin treatment. CTCs were then examined using a Microfluidic Chip device. CTCs positive for vimentin and pan-cytokeratin were defined as mesenchymal and epithelial CTCs, respectively. Progression-free survival (PFS) and clinical response were assessable in 20 and 18 patients, respectively, in relation to the number of CTCs. RESULTS: Numbers of total CTCs were significantly increased in patients with progressive disease during treatment (p = 0.006). Median PFS was 14.6 weeks and patients with more total and mesenchymal CTCs at baseline had significantly shorter PFS (p = 0.0013 and 0.013, respectively). Multivariate logistic regression analysis revealed small number of total baseline CTCs and long disease-free survival to be related to long PFS (p = 0.0004 and 0.020, respectively). CONCLUSIONS: Our data suggest that determining both mesenchymal and epithelial CTCs at baseline might be a good tool for predicting eribulin responsiveness. Evaluation of mesenchymal CTC can be considered as a parameter in larger studies, while most clinical trials are currently employing only the detection of the epithelial cellular adhesion molecule (EpCAM).

Differences in Ki67 expressions between pre- and post-neoadjuvant chemotherapy specimens might predict early recurrence of breast cancer.

The prognosis of breast cancer patients not obtaining a pathological complete response (pCR) with neoadjuvant chemotherapy (NAC) is poorer than that of pCR patients. Identifying new prognostic factors for non-pCR patients is important because fractions of this population might benefit from novel adjuvant treatments currently under development. High Ki67 expression in remnant disease after NAC has been described as a poor prognostic factor. Studies have shown that a reduction in Ki67 expression is more often observed in good responders to chemotherapy. We hypothesized that the change in Ki67 expression might be useful for predicting patient outcomes and thus retrospectively examined pairs of biopsy and surgical specimens of breast tissue from individual patients. One hundred sixteen patients with remnant invasive disease in the breast, who received NAC and underwent surgery at our institution, were retrospectively examined. Differences in Ki67 expression between pre- and post-NAC specimens were analyzed in relation to patient outcomes. The mean Ki67 expression value after NAC was higher in patients who developed metastasis than in those without metastasis (P<.01). Tumors showing higher Ki67 expression in the surgical than in the biopsy specimen were more frequent in patients with metastasis (P<.01). This trend was more obvious in patients who developed metastasis within 1 year after surgery. Our results indicate that a difference in Ki67 expressions after versus before NAC might be an important predictor of early metastasis. Evaluating not only absolute Ki67 values, but also any changes in response to NAC, may improve the prediction of patient outcomes.

Combination of Cancer Stem Cell Markers CD44 and CD24 Is Superior to ALDH1 as a Prognostic Indicator in Breast Cancer Patients with Distant Metastases.

The combination of CD44 and CD24, or aldehyde dehydrogenase 1 (ALDH1) alone, is a widely used cancer stem cell marker in breast cancer. However, no conclusion has yet been reached as to which marker is the best for characterizing cancer stemness. Immunohistochemical evaluation using cancer stem cell markers is clearly less common clinically than in basic experiments and how the expressions of these markers relate to patient outcomes remains controversial. To investigate whether combining these markers might improve the prediction of patient outcomes, we immunohistochemically examined clinical samples. Primary invasive breast cancer samples from 61 patients who eventually developed distant metastases after curative surgery were immunohistochemically examined. All patients were free of metastatic disease at the time of surgery and received standard adjuvant systemic treatments. CD44+/24- and ALDH1-positive rates in primary tumors differed according to intrinsic subtype. ER-positive patients with CD44+/24- tumors had significantly longer disease-free-survival than all other ER-positive patients (p = 0.0047). On the other hand, CD44+/24- tumors were associated with poor outcomes of ER-negative patients (p = 0.038). Finally, expression patterns of CD44 and ALDH1 in single tumors were strikingly different and there were virtually no individual double-stained cells. Thus, this combination does not allow evaluation of relationships with patient outcomes. Our results raise the possibility of CD44+/24- being a good prognostic marker, one which would allow treatment effects and outcomes to be predicted in patients with recurrent breast cancer.

Activated Caspase 3 Expression in Remnant Disease After Neoadjuvant Chemotherapy May Predict Outcomes of Breast Cancer Patients.

BACKGROUND: A number of studies have indicated that patients obtaining a pathological complete response (pCR) from neoadjuvant chemotherapy (NAC) have a good prognosis; however, prognostic factors for non-pCR patients are not yet well-established. By examining remnant cancer in non-pCR patients, the expression of cleaved Caspase 3 (Casp3), an activated apoptotic marker, was immunohistochemically investigated to determine whether this protein has the potential to serve as a novel marker for predicting patient outcomes. METHODS: We investigated 218 patients with invasive breast cancer who received NAC and underwent surgery during the 2006 through 2008 period at our institution. Following surgery, standard adjuvant endocrine therapy was administered if a tumor was hormone receptor-positive. Casp3 was evaluated in remnant cancer based on the number of positive cells in five high-power fields. RESULTS: pCR was obtained in 49 patients, and 50 of the 169 non-pCR patients developed recurrences during the median 82-month observation period. We found large tumor size, lymph node involvement, lymph vessel invasion, estrogen receptor-negative, progesterone receptor-negative, high Ki67 and high Casp3 expression to be factors related to tumor recurrence. A logistic regression model revealed that lymph node involvement, as well as high Ki67 and Casp3, to be factors independently predicting recurrence, while lymph vessel invasion and high Ki67 expression were found to be related to breast cancer mortality. CONCLUSIONS: Patients with remnant cancer showing high Casp3 expression had poor outcomes. Our results showed that Casp3 is a potential prognostic marker for non-pCR patients.

Ki67 Heterogeneity in Estrogen Receptor-Positive Breast Cancers: Which Tumor Type Has the Most Heterogeneity?

Heterogeneity of Ki67 expression, often seen in breast cancer, can make evaluation of the expression of this marker difficult and give rise to confusion when considering adjuvant treatments for patients. Herein, we investigated estrogen receptor-positive breast cancers to reveal the tumor characteristics associated with Ki67 heterogeneity. Surgical specimens from 85 invasive ductal carcinomas of no special type and 13 invasive lobular carcinomas were examined. We first calculated the differences between Ki67 expression in a hot spot and those in 4 random fields on the same slide. We then evaluated Ki67 heterogeneity within the tumor, based on these differences. Among clinicopathological factors, solid-tubular carcinoma, an architectural growth pattern subtype of invasive ductal carcinoma, correlated with high Ki67 heterogeneity (P < .05). Our results indicate that we might need to be aware of histological patterns when selecting appropriate microscopic fields for evaluating Ki67 expression.

Application of a 70-Gene Expression Profile to Japanese Breast Cancer Patients.

BACKGROUND: As data on using MammaPrint®, a 70-gene expression profile for molecular subtyping of breast cancer, are limited in Japanese patients, we aimed to determine the gene profiles of Japanese patients using MammaPrint and to investigate its possible clinical application for selecting adjuvant treatments. PATIENTS AND METHODS: 50 women treated surgically at our institution were examined. The MammaPrint results were compared with the St Gallen 2007 and intrinsic subtype risk categorizations. RESULTS: Of 38 cases judged to be at intermediate risk based on the St Gallen 2007 Consensus, 11 (29%) were in the high-risk group based on MammaPrint. 1 of the 30 luminal A-like tumors (3%) was judged as high risk based on MammaPrint results, whereas 7 of the 20 tumors (35%) categorized as luminal B-like or triple negative were in the low-risk group. There have been no recurrences to date in the MammaPrint group, and this is possibly attributable to most of the high-risk patients receiving chemotherapy that had been recommended on the basis of their MammaPrint results. CONCLUSIONS: Our results indicate that MammaPrint is applicable to Japanese patients and that it is of potential value in current clinical practice for devising individualized treatments.

Menstrual cycle could affect Ki67 expression in estrogen receptor-positive breast cancer patients.

BACKGROUND: Ki67 is a potent prognostic marker for determining systemic treatment of patients with hormone receptor-positive breast cancer. However, evaluation of Ki67 expression can be difficult, due mostly to its heterogeneity. The Ki67 expression level, which indicates that a cell is undergoing division (cell cycle), rises when proliferation activity increases. Thus, Ki67 expression might be affected by hormonal stimuli. We hypothesised that Ki67 expression level might change during the menstrual cycle. We examined pairs of biopsy and surgical specimens from individual patients to evaluate this hypothesis. METHODS: First, the effects of estradiol on Ki67 expression in breast cancer cell lines were examined employing immunocytochemistry and Western blotting. Next, differences in Ki67 expression between biopsy and surgical specimens from 131 patients with estrogen receptor-positive tumours were retrospectively examined. RESULTS: In vitro experiments showed Ki67 expression in estrogen receptor-positive cancer cells to be dependent on estradiol stimulation. Ki67 expression was higher in biopsy samples collected in the luteal phase than in those from other phases. When biopsy and surgical samples were obtained at different times during the menstrual cycle in the same individual, there were differences in Ki67 expression between these samples. Those collected in the luteal phase showed higher Ki67 expression than samples obtained during other phases (p<0.01). CONCLUSIONS: Ki67 expression varied in the same patients according to menstrual cycle phase. Our results suggest that Ki67 expression in estrogen receptor-positive breast cancer should be carefully assessed bearing in mind the patient's menstrual cycle, since the interpretation of expression could affect treatment decisions.

Protein expression and methylation of DNA repair genes hMLH1, hMSH2, MGMT and BRCA1 and their correlation with clinicopathological parameters and prognosis in basal-like breast cancer.

AIMS: Basal-like breast cancer (BLBC) is characterized by aggressive behaviour; its genesis is the perturbation of DNA repair as a consequence of BRCA1 methylation or mutation. We comparatively evaluated alterations of DNA repair proteins and p53 between BLBC and non-BLBC cases. METHODS AND RESULTS: Tumour sections from 104 BLBC and 89 non-BLBC patients were immunostained for hMLH1, hMSH2, MGMT, BRCA1 and p53. Methylation status of DNA repair genes was analysed by methylation-specific PCR, and p53 mutation was examined by direct sequencing. Immunoreactive levels of hMLH1 and MGMT were lower in BLBC, whereas the levels of hMSH2 and p53 were higher, compared to non-BLBC (P ≤ 0.014). Reduced expression of hMLH1 [hazard ratio (HR) 5.26, P = 0.001] and preserved expression of MGMT (HR 2.58, P = 0.039) proved to be independent predictors of poor survival in BLBC patients. DNA repair genes were methylated in approximately 20-40% of BLBCs without a significant relationship between their methylation and p53 mutation. BRCA1 methylation was associated with the loss of its protein expression (P = 0.004). MGMT methylation was linked to larger tumour size (P < 0.001). CONCLUSIONS: Perturbations of the DNA repair system might be different between BLBC and non-BLBC. Alterations of hMLH1 and MGMT appear important for tumour progression and survival in BLBC patients.