Masafumi Shimoda

Sox17 plays a substantial role in late-stage differentiation of the extraembryonic endoderm in vitro

Sox17 is a Sry-related HMG-box transcription factor developmentally expressed in both the definitive endoderm and extraembryonic endoderm (ExE). Although Sox17–/– mouse embryos have a defective definitive gut endoderm, their developing ExE is morphologically intact. Here, we aimed to investigate the role of Sox17 in ExE development by using an in vitro differentiation system of embryonic stem cells (ESCs). Although forced Sox17 expression in ESCs did not affect ExE commitment, it facilitated the differentiation of ESC-derived primitive endoderm cells into visceral and parietal endoderm cells. This event was inhibited by the forced expression of Nanog, a negative regulator of differentiation of ESCs into the ExE. Although Sox17–/– ESCs could differentiate into primitive endoderm cells, further differentiation was severely impaired. These results indicate a substantial involvement of Sox17 in the late stage of ExE differentiation in vitro. Furthermore, the expression of Sox7 – another Sox factor, concomitantly expressed with Sox17 in the developing ExE – was suppressed during the in vitro differentiation of Sox17–/– ESCs, but it was maintained at a high level in the extraembryonic tissues of Sox17–/– embryos. These findings possibly explain the discrepancy between the ExE phenotype derived from Sox17–/– ESCs and that of Sox17–/– embryos.

Mutational analysis of MED12 in fibroadenomas and phyllodes tumors of the breast by means of targeted next-generation sequencing

We aimed to analyze MED12 mutation in fibroadenomas (FAs) and phyllodes tumors (PTs) of the breast, which are closely related and consist of epithelial and stromal components. Targeted deep-sequencing using next-generation sequencing was performed in FAs (n = 58) and PTs (n = 27). The frequency of MED12 mutant tumors was significantly higher (P = 0.016) in PTs (74.1 %) than in FAs. (46.6 %). As for FAs, this frequency was significantly higher (P = 0.001) for intracanalicular type (69.0 %) than for other histological subtypes such as pericanalicular, organoid, and mastopathic types (24.1 %). Laser microdissection study revealed that stromal cells, but not epithelial cells, harbored MED12 mutations in both FAs and PTs. MED12 mutation is implicated in the pathogenesis of both FAs and PTs. The similarly high frequency of MED12 mutation in intracanalicular type FAs suggests that they are most closely related to PTs. It is thus speculated that FAs with MED12 mutation are more likely to progress to PTs.

Utilization of autologous vein graft for replacement of the inferior vena cava in living-donor liver transplantation for obliterative hepatocavopathy

Obliterative hepatocavopathy (OHC) is a subtype of Budd‐Chiari syndrome in which stenosis or obstruction of the retrohepatic inferior vena cava (IVC) is observed. Although IVC replacement is necessary in OHC patients, there are hardly any graft vessels available for IVC reconstruction during living‐donor liver transplantation (LDLT). Here, we describe a novel technique of IVC reconstruction using only the autologous blood vessels in an OHC patient during LDLT. In this case, sufficient drainage of the hepatic outflow and reconstruction of the venous return from the lower half of the body were simultaneously required. Therefore, we substituted the retrohepatic IVC with the suprarenal IVC of the recipient, and we reconstructed the IVC continuity by using the autologous internal jugular vein and external iliac vein. The operation was safe, and the postoperative venous drainage from the hepatic tributaries was in good condition. This procedure might be an option for IVC replacement during LDLT.

BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer

The breast cancer susceptibility gene 1 (BRCA1) and glutathione S‐transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation‐specific PCR assay. Patients with BRCA1‐methylated (n = 15) or BRCA1‐unmethylated (n = 15) tumors and those with GSTP1‐methylated (n = 9) or GSTP1‐unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro‐dissected normal epithelial cells from the formalin‐fixed paraffin‐embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1‐methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1‐unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic‐activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1‐methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation.

Methylated DNA and high total DNA levels in the serum of patients with breast cancer following neoadjuvant chemotherapy are predictive of a poor prognosis

In a previous study, we established a one-step methylation-specific polymerase chain reaction (OS-MSP) assay for the detection of methylated DNA (met-DNA) and total DNA levels in serum. For the present study, this OS-MSP assay was used for patients with breast cancer treated with neoadjuvant chemotherapy (NAC) in order to investigate the prognostic significance of met-DNA and total DNA levels. Following treatment with NAC and prior to surgery, serum samples obtained from 120 patients with stage II/III breast cancer were subjected to the OS-MSP assay for analysis of the glutathione S-transferase pi 1, Ras association (RalGDS/AF-6) domain family member 1 and retinoic acid receptor β2 genes. The detection of methylation in a minimum of one of these genes indicated a positive outcome of the assay. The total DNA content of the serum was also determined. Of the 120 stage II/III patients, seven (6%) were positive for met-DNA in serum and showed a significantly worse overall survival (OS) time compared with patients negative for met-DNA (n=113) (5-year OS, 43 vs. 85%; P=0.002). The patients with high total DNA levels in serum (n=40) also showed a significantly worse OS compared with those with low total DNA levels (n=80) (65 vs. 91%; P<0.001). The presence of met-DNA and high total DNA levels in the serum were found to be significant prognostic factors that are independent of a pathological complete response by multivariate analysis. Following NAC, met-DNA and high total DNA levels in the serum detected with the OS-MSP assay constitute novel prognostic factors for patients with breast cancer; this may be clinically useful for the prognosis prediction for patients who do not achieve a pathological complete response following NAC.

Clinicopathological analysis of breast ductal carcinoma in situ with ALDH1-positive cancer stem cells.

Objective: The aim of this study was to elucidate the clinicopathological characteristics of breast ductal carcinomas in situ (DCIS) with aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cells (CSCs). Methods: DCIS (n = 194) were subjected to immunohistochemical staining and results were examined for associations with various clinicopathological parameters. The ALDEFLUOR assay for breast cancer cell lines was also performed to determine the proportion of CSCs according to intrinsic subtype. Results: DCIS with ALDH1-positive CSCs were significantly (p < 0.05) more likely to be estrogen receptor-α (ER)-negative, progesterone receptor (PgR)-negative, Ki67-positive and HER2-positive tumors. Luminal A subtype (8.6%) showed a significantly (p < 0.001) lower ALDH1 positivity than the other subtypes [luminal B (50.0%), luminal HER2 (36.8%), HER2 (35.3%) and triple-negative subtype (26.7%)]. Double immunostaining revealed that ALDH1-positive CSCs did not overlap with ER-, PgR- or Ki67-positive tumor cells but did overlap with HER2-positive tumor cells. The percentage of ALDEFLUOR-positive CSCs was lower in the luminal A cell lines (0.02% for T-47D and 0.4% for MCF7) than in the luminal HER2 (9.1% for BT-474 and 9.5% for MDA-MB-361), HER2 (13.9% for AU565 and 33.2% for SK-BR-3) and triple-negative cell lines (28.4% for MDA-MB-231 and 30.7% for MDA-MB-468). Conclusions: Our results suggest that most CSCs in DCIS are in the G0 phase (Ki67 negative) and do not express ER or PgR, but they do express HER2 in HER2-positive tumors.

One-step nucleic acid amplification assay for intraoperative prediction of non-sentinel lymph node metastasis in breast cancer patients with sentinel lymph node metastasis

The aim of the present study was to construct the intra-operative prediction model of non-sentinel lymph node (non-SLN) metastasis in breast cancer patients with SLN metastasis using one-step nucleic acid amplification (OSNA). Of 833 breast cancer patients (T1-T2, N0) who underwent SLN biopsy and had their SLNs examined intra-operatively with the OSNA assay, 161 with SLN metastasis and treated with completion axillary lymph node dissection (cALND) were randomly divided into a training (n = 81) and a validation (n = 80) cohort. Non-SLN metastasis of the training cohort was associated with the number of positive SLNs (P = 0.001), CK19 mRNA copy number (P = 0.001), and clinical tumor size (P = 0.055). These parameters were used to construct the intra-operative prediction model of non-SLN metastasis. Its diagnostic accuracy (AUC of ROC curve) was 0.809 and 0.704 for the training and validation cohorts, respectively. The intra-operative prediction model using OSNA may have a diagnostic accuracy of non-SLN metastasis comparable to that of the conventional, post-operative prediction model, indicating that it might help decide the indication for cALND.

Correlation of Methylated Circulating Tumor DNA With Response to Neoadjuvant Chemotherapy in Breast Cancer Patients.

Micro‐Abstract Correlation of methylated circulating tumor DNA (met‐ctDNA) with tumor response to neoadjuvant chemotherapy (NAC) was evaluated in breast cancer patients. In patients with positive met‐ctDNA before NAC, met‐ctDNA significantly correlated with tumor response and was found to be a more sensitive marker than carcinoembryonic antigen and cancer‐associated antigen 15‐3. The possibility has also been suggested that met‐ctDNA might be useful in monitoring the postoperative recurrence. Background: Circulating tumor DNA (ctDNA) is known to harbor tumor‐specific genetic or epigenetic alterations. In the present study, the correlation of ctDNA with tumor response to neoadjuvant chemotherapy (NAC) was evaluated in primary breast cancer patients. Patients and Methods: Plasma samples were obtained from 87 primary breast cancer patients (stage II‐III) before and after NAC, as well as 1 year after surgery. Methylated ctDNA (met‐ctDNA) was determined by one‐step methylation‐specific PCR (OS‐MSP) for the promoter region of RASSF1A. Results: The positivity (23.0%, 20/87) of met‐ctDNA before NAC was significantly (P < .05) higher than that of carcinoembryonic antigen (CEA) (8.6%) and cancer‐associated antigen (CA) 15‐3 (7.4%). In the patients with positive met‐ctDNA before NAC, met‐ctDNA significantly decreased after NAC in those with disease that responded to therapy (P = .006), but not in patients whose disease did not respond to therapy. Met‐ctDNA after NAC was found to be significantly (P = .008) correlated to the extent of residual tumor burden. Of the 7 patients who showed an increase in met‐ctDNA at 1 year after surgery, 3 developed recurrence. Conclusion: Met‐ctDNA is a more sensitive marker than CEA and CA15‐3, and it might be useful in monitoring the clinical tumor response to NAC. In addition, the potential use of met‐ctDNA as a tumor marker for monitoring postoperative recurrence has been suggested.

Prediction of pathological complete response to neoadjuvant chemotherapy by magnetic resonance imaging in breast cancer patients

The purpose of this study was to evaluate whether the baseline breast MRI findings would be useful for the prediction for pathological complete response (pCR) by breast cancer patients to neoadjuvant chemotherapy. Primary breast cancer patients (stage II-III) preoperatively treated with sequential paclitaxel (12 cycles) and fluorouracil, epirubicin, and cyclophosphamide (4 cycles), followed by surgery were retrospectively enrolled, and 229 patients were eligible. Before chemotherapy, breast MRI studies were performed. Breast tumors were dichotomized into round + oval and irregular types based on MRI morphology. The round + oval tumors showed a significantly higher pCR rate than the irregular tumors (42.0% vs 17.3%; P < 0.001). In addition, PAM50 analysis revealed that basal and HER2-enriched tumors were significantly more prevalent among round + oval than irregular type tumors (P = 0.015). Baseline MRI morphology appears to be a significant predictor for pCR. The higher rate of the basal and HER2-enriched tumors among the round + oval tumors may explain their better chemo-sensitivity.

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing

PurposeLiquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations.MethodsWhole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected.ResultsWe identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways.ConclusionsAlthough the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.