Keiichi Murakami

Insulin-like growth factor-1 receptor gene expression is associated with survival in breast cancer: a comprehensive analysis of gene copy number, mRNA and protein expression

Insulin-like growth factor-1 receptor (IGF1R) plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. In a series of 325 primary invasive breast cancer patients, we performed a comprehensive analysis of IGF1R at the levels of gene copy number, mRNA expression and protein expression. The relationship between the IGF1R status and the clinicopathological characteristics and prognosis was evaluated. IGF1R mRNA levels not only correlated with protein expression, but also were significantly associated with several clinicopathological parameters and prognosis. Patients with low nuclear grade, negative axillary lymph nodes, positive hormone receptor, negative Her2, negative Ki67, and luminal subtype tumors showed higher expression levels of IGF1R mRNA, which was shown to be a significant univariate parameter for both relapse-free survival and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for BCSS. IGF1R protein expression showed an association with a prolonged BCSS in univariate analysis. In contrast, IGF1R gene copy number was not correlated with mRNA and protein expression, and harbored no prognostic value. When studied in the luminal tumor subtype groups, IGF1R mRNA level was still significantly associated with a better BCSS. Overall, our data indicated a correlation between IGF1R mRNA expression and protein expression in primary breast cancer. In particular, IGF1R mRNA expression appeared to be a good prognostic marker both in the entire cohort and in the luminal subtype group. These data may serve as background information for IGF1R-targeted therapy.

High survivin mRNA expression is a predictor of poor prognosis in breast cancer: A comparative study at the mRNA and protein level

BackgroundSurvivin plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. The purpose of this study was to examine the expression of survivin and its role in predicting clinical outcome in a series of human breast cancer cases both at the mRNA and protein level.MethodsFormalin-fixed paraffin-embedded tumor tissues from 245 female patients with invasive breast cancer and 13 patients with ductal carcinoma in situ were examined for survivin mRNA by quantitative real-time RT-PCR (RT-qPCR). In addition, 237 of these tumors with invasive breast cancer were available for immunohistochemistry (IHC). The relationship between survivin status and clinicopathological characteristics and prognosis was evaluated.ResultsRT-qPCR revealed that high levels of survivin mRNA were strongly associated with high nuclear grade, positive axillary lymph nodes, negative hormone receptor status, positive Her2 amplification, higher Ki67 labeling index, and presence of vascular invasion. In the Cox proportional regression model analysis, survivin mRNA was shown to be a significant univariate parameter for relapse-free survival (RFS), distant relapse-free survival (DRFS), and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for RFS, DRFS, and BCSS. In hormone receptor (HR)-positive/Her2-negative subtype cases, survivin mRNA expression was also an independent predictor in terms of DRFS. Immunohistochemically, positive staining was seen in the cytoplasm and/or nucleus of cancer cells, although this did not correlate with the mRNA level, and harbored no prognostic value.ConclusionsHigh mRNA expression of survivin was an independent marker of poor prognosis both in the entire cohort and in the HR-positive/Her2-negative subtype, whereas the protein expression of survivin was not. These findings suggest that RT-qPCR can provide more reliable data than IHC in validating the prognostic significance of survivin for breast cancer patients.

Clinical significance of pretherapeutic Ki67 as a predictive parameter for response to neoadjuvant chemotherapy in breast cancer; is it equally useful across tumor subtypes?

BACKGROUND Ki67 has been identified as a prognostic and predictive marker for breast cancer and it was suggested that it may contribute to pathologic complete response (pCR) after neoadjuvant chemotherapy. It is unclear whether expression of Ki67 is particularly helpful for prediction of pCR across tumor subtypes. METHODS Pretherapeutic Ki67 was evaluated in a series of 121 breast cancer core biopsies. After neoadjuvant chemotherapy, we used postoperative specimens to evaluate the pCR status. Several parameters predictive of pCR were identified using logistic regression analysis. We investigated subgroups defined by estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2, in which predicting pCR with Ki67 might be feasible. RESULTS Ki67 was found to be an independent predictor of pCR in multivariate analysis (odds ratio [OR], 3.62; 95% CI, 1.21-10.8). When stratified by ER, the above significance was exclusive to ER-positive tumors (OR, 6.24; 95% CI, 1.40-27.7). Using an receiver-operating characteristic curve, we obtained moderate discriminative accuracy with an area under the curve of 0.7752 for Ki67 prediction of pCR in ER-positive tumors. In subgroup analysis, patients with high Ki67 showed significantly improved pCR rate in luminal-type disease, with a median Ki67 value of 43% in the patients who achieved pCR, versus 29% for those without pCR (P = .018), whereas no associations were observed in other subtypes. CONCLUSION Our results suggest that stratification according to Ki67 levels might improve predictive significance of the response in hormone-responsive breast cancer. Even in these subtypes assumed to be less chemosensitive, some patients with highly proliferative tumors derive a significant benefit from chemotherapy, and consequently it is important to identify them.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients

BACKGROUND The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. METHODS The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. RESULTS cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. CONCLUSIONS We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.Background The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. Methods The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. Results cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. Conclusions We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients

BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). RESULTS: Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.

Clinical significance of plasma cell-free DNA mutations in PIK3CA , AKT1 , and ESR1 gene according to treatment lines in ER-positive breast cancer

The somatic activation of PI3K/AKT pathway mutations, PIK3CA and AKT1, and ESR1 mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive procedure to quickly assess and monitor disease progression or therapeutic effect in breast cancer (BC) patients, but the clinical significance of these mutations in late treatment lines (TLs) remains unclear. The subjects of this study were a total of 251 plasma samples from 128 estrogen receptor-positive (ER+) BC patients. Of these plasma samples, 133 were from 73 primary BC (PBC) patients, and 118 plasma samples were from 68 metastatic BC (MBC) patients. We developed droplet digital PCR (ddPCR) assays to verify the clinical significance of PIK3CA, AKT1, and ESR1 mutations in these patients. cfDNA PIK3CA mutations were observed in 15.1% of the PBC patients, while a cfDNA AKT1 mutation was observed in 1.4% of patients, and cfDNA ESR1 mutations were observed in 2.7% of patients. Patients with detectable cfDNA PIK3CA mutations were not associated with clinical outcomes. According to the TL, the prevalence of the PIK3CA and ESR1 mutations in cfDNA were lower in early TLs compared with late TLs. In the early TL group, patients with cfDNA PIK3CA mutations had a shorter time to treatment failure (TTF) than patients without mutations (P = 0.035). However, there was no statistically significant difference between patients with or without cfDNA ESR1 mutations. However, in the late TL group, patients with cfDNA ESR1 mutations had a shorter TTF than patients without mutations (P = 0.048). However, there was no statistically significant difference between patients with or without cfDNA PIK3CA mutations. Since the prevalence of cfDNA AKT1 mutation is low in both PBC and MBC patients, the impact of AKT1 mutations on the prognosis remains unclear. We have demonstrated the difference in the clinical significance of the hotspot PIK3CA, AKT1, and ESR1 mutations in cfDNA for each TL in ER+ BC patients.

Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients

BackgroundESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations.MethodsWe screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations (E380Q, Y537S, Y537N, Y537C, and D538G) using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated.ResultsWe detected ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment.ConclusionsWe have shown that there are distinct populations of ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.

Prospective observational trial of re-antiestrogen therapy after becoming resistant to ethinylestradiol treatment in hormone-dependent metastatic breast cancer.

161 Background: After heavily treated estrogen deprivation therapy by aromatase inhibitors (AIs), an additional estrogen therapy may be useful in a certain condition. Furthermore, the estrogen therapy might sensitize a subsequent anti-estrogen therapy after the acquired resistance to estrogen. METHODS In the patients who obtained disease control (SD or more) by ethinylestradiol (EE2) therapy (1.5-3mg TID), the efficacy of anti-estrogen therapy using AI or fulvestrant was examined as a subsequent endocrine therapy. Primary endpoint was clinical benefit rate (CBR), and secondary endpoints were response rate (RR) and time to treatment failure (TTF). (Registration; UMIN000002831, prospective observational trial, sub-protocol analysis) Results: EE2 therapy was performed in 22 of postmenopausal breast cancer patients with ER-positive HER2-negative in primary or recurrent tumor, who had heavily pre-treated by endocrine therapies, including chemotherapies. At the median observation period of 13.6 months, RR of 41% (9/22) and CBR of 50% (11/22) were obtained (CR; 0, PR; 9, long SD; 2, SD; 4, PD; 3) and 4 patients withdrew this trial due to adverse events of early endocrine related symptoms and cerebral infarction. Although two patients were still ongoing, the median of duration of disease-control was 46 weeks (range; 23-62+). In thirteen patients who obtained disease control (SD or more) by EE2 therapy, fulvestrant for 6 patients or AI for 7 patients was used as a subsequent therapy. Fifty percent of fulvestrant group (3/6) and 43% of AI group (3/7) showed clinical benefit (PR or long SD). TTF of fulvestrant group was 15 weeks (median; range; 5-55+) and that of AI group was 19 weeks (median; range 5-33+). Furthermore, two patients who were responders to previous AI, got long SD to the subsequent EE2 re-treatment. CONCLUSIONS EE2 therapy after resistance to estrogen deprivation therapy could be very useful, and a subsequent anti-estrogen therapy by AI or fulvestrant could be also effective in a certain condition. Thus, the changes of the endocrine environment by EE2 may induce the sensitization to hormone dependency in acquired hormone-resistant tumors. CLINICAL TRIAL INFORMATION 000002831.