Yasuto Naoi

GSTP1 expression predicts poor pathological complete response to neoadjuvant chemotherapy in ER-negative breast cancer

The purpose of the present study was to investigate the association of glutathione S‐transferase P1 (GSTP1) expression with resistance to neoadjuvant paclitaxel followed by 5‐fluorouracil/epirubicin/cyclophosphamide (P‐FEC) in human breast cancers. The relationship of GSTP1 expression and GSTP1 promoter hypermethylation with intrinsic subtypes was also investigated. In this study, primary breast cancer patients (n = 123, stage II–III) treated with neoadjuvant P‐FEC were analyzed. Tumor samples were obtained by vacuum‐assisted core biopsy before P‐FEC. GSTP1 expression was determined using immunohistochemistry, GSTP1 promoter methylation index (MI) using bisulfite methylation assay and intrinsic subtypes using DNA microarray. The pathological complete response (pCR) rate was significantly higher in GSTP1‐negative tumors (80.0%) than GSTP1‐positive tumors (30.6%) (P = 0.009) among estrogen receptor (ER)‐negative tumors but not among ER‐positive tumors (P = 0.267). Multivariate analysis showed that GSTP1 was the only predictive factor for pCR (P = 0.013) among ER‐negative tumors. Luminal A, luminal B and HER2‐enriched tumors showed a significantly lower GSTP1 positivity than basal‐like tumors (P = 0.002, P < 0.001 and P = 0.009, respectively), while luminal A, luminal B and HER2‐enriched tumors showed a higher GSTP1 MI than basal‐like tumors (P = 0.076, P < 0.001 and P < 0.001, respectively). In conclusion, these results suggest the possibility that GSTP1 expression can predict pathological response to P‐FEC in ER‐negative tumors but not in ER‐positive tumors. Additionally, GSTP1 promoter hypermethylation might be implicated more importantly in the pathogenesis of luminal A, luminal B and HER2‐enriched tumors than basal‐like tumors. (Cancer Sci 2012; 103: 913–920)

Prediction of pathologic complete response to sequential paclitaxel and 5-fluorouracil/epirubicin/cyclophosphamide therapy using a 70-gene classifier for breast cancers.

Sequential administration of paclitaxel plus combined fluorouracil, epirubicin, and cyclophosphamide (P‐FEC) is 1 of the most common neoadjuvant chemotherapies for patients with primary breast cancer and produces pathologic complete response (pCR) rates of 20% to 30%. However, a predictor of pCR to this chemotherapy has yet to be developed. The authors developed such a predictor by using a proprietary DNA microarray for gene expression analysis of breast tumor tissues.

Clinicopathological analysis of GATA3-positive breast cancers with special reference to response to neoadjuvant chemotherapy

BACKGROUND The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3 (GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy. PATIENTS AND METHODS Tumor specimens obtained before neoadjuvant chemotherapy [paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic subtyping. RESULTS Seventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were significantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR) was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. multivariate analysis showed that tumor size, human epidermal growth factor receptor 2 (her2), and gata3 were independent predictors of pcr. CONCLUSIONS GATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and were mostly classified as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor response to chemotherapy.

Gene expression signature of TP53 but not its mutation status predicts response to sequential paclitaxel and 5-FU/epirubicin/cyclophosphamide in human breast cancer

PURPOSE The aim of this study was to determine whether TP53 mutation status (MS) can predict response of breast cancer to paclitaxel followed by 5-FU/epirubicin/cyclophosphamide (P-FEC). TP53 gene expression signature (GES) was also examined for its predictive capability of response to P-FEC since TP53 GES provides a more accurate measure of the functional configuration of TP53. METHODS Tumor samples were obtained from 72 primary breast cancer patients (stage II/III) before neoadjuvant chemotherapy (P-FEC) and analyzed for identification of TP53 MS (genomic sequencing), TP53 GES (DNA microarray), and p53 protein expression (immunohistochemistry). RESULTS Of 72 breast tumors, 16 were TP53 mutant-type (TP53 mt) and 56 were wild-type (TP53 wt). 29 tumors (40%) were positive for p53 protein by immunohistochemistry. DNA microarray analysis showed that 27 were TP53 mt-like tumors and 45 were TP53 wt-like tumors, depending on the expression signature of the TP53-related 31-genes. There was no statistically significant difference in pathological complete response (pCR) rates between TP53 mt and wt tumors (19% vs 23%) and between p53 positive and negative tumors (24% vs 21%) but TP53 mt-like tumors showed a significantly (P=0.019) higher pCR rate (37%) than TP53 wt-like tumors (13%) (Hazard ratio, 3.82; 95% C.I., 1.20-12.21). CONCLUSIONS TP53 GES, but not TP53 MS and p53 protein expression, is predictive of response to neoadjuvant P-FEC, suggesting that TP53 GES more correctly reflects the functionality of TP53.

Connexin26 expression is associated with aggressive phenotype in human papillary and follicular thyroid cancers.

Connexin26 (Cx26), a component of GAP junctions and until recently believed to be a tumor suppressor gene, has been shown to play an important role in lymphatic invasion as well as lymph node and distant metastases in squamous lung cancer and breast cancer. In the study presented here, we investigated Cx26 expression in human papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) and its relationship with various clinicopathological parameters. Of 69 PTCs, 33 were positive for Cx26 (47.8%), as were five of 11 FTCs (45.5%), all follicular thyroid adenomas (n=22) and normal thyroid tissues (n=20) were negative for Cx26. A statistically significant association was observed between Cx26 expression and large tumor size (p=0.028 for PTC) and lymph node metastases (p=0.053 (marginally significant) for PTC and p=0.035 for FTC). Presence of intra-glandular dissemination of tumor cells was significantly (p=0.048) more frequent in Cx26-positive (30.3%) than Cx26-negative PTCs (11.1%). Lymphatic vessel invasion was more frequent in Cx26-positive PTCs (6.1%) than in Cx26-negative PTCs (0%) though the difference was not statistically significant. These results suggest that Cx26 may be implicated in the pathogenesis of PTC and FTC and is associated with the biologically aggressive phenotypes of these tumors.

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.

Construction of novel immune-related signature for prediction of pathological complete response to neoadjuvant chemotherapy in human breast cancer

BACKGROUND The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data. PATIENTS AND METHODS DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets. RESULTS The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09). CONCLUSION The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

Association between c-myc amplification and pathological complete response to neoadjuvant chemotherapy in breast cancer

BACKGROUND The aim of this study was to investigate whether c-myc amplification in human breast cancer is associated with response to neoadjuvant chemotherapy comprising paclitaxel followed by 5-FU/epirubicin/cyclophosphamide (P-FEC). METHODS Tumour tissue samples were obtained before neoadjuvant chemotherapy (P-FEC) from 100 primary breast cancer patients (stage II/III). C-myc and HER2 amplification were examined by FISH, and oestrogen receptor (ER), progesterone receptor (PR), Ki67, and topoisomerase 2α (TOP2A) expression were examined immunohistochemically. Pathological complete response (pCR) was defined by a complete loss of tumour cells in the breast without any lymph node metastasis. RESULTS C-myc amplification was observed in 40% (40/100) of breast tumours, and was significantly associated with ER-negative tumours (23/40 for ER(-) versus 17/60 for ER(+), P=0.004), high histological grade tumours (11/18 for grade 3 versus 29/82 for grades 1+2, P=0.043) and TOP2A-positive tumours (28/51 for TOP2A(+) versus 12/49 for TOP2A(-), P=0.002). pCR rate was 20% for total patients (10.0% for ER(+) and 35.0% for ER(-)). Further, breast tumours with c-myc amplification (c-myc(+)) showed a significantly (P=0.041) higher pCR rate (12/40) than those without such amplification (c-myc(-)) (8/60). This association between pCR and c-myc amplification was observed in ER-positive tumours (4/17 for c-myc(+) versus 2/43 for c-myc(-), P=0.048) but not in ER-negative tumours (8/23 for c-myc(+) versus 6/17 for c-myc(-), P=0.973). CONCLUSION Our results suggest that c-myc amplification is significantly associated with a high pCR rate to P-FEC in breast tumours, especially in ER-positive tumours.

High genomic grade index associated with poor prognosis for lymph node‐negative and estrogen receptor‐positive breast cancers and with good response to chemotherapy

The aim of the present study was to investigate the prognostic value of the genomic grade index for lymph node‐negative and estrogen receptor (ER)‐positive breast cancers of Japanese women treated with adjuvant hormonal therapy alone, as well as the relation between genomic grade index and pathological complete response (CR) to neoadjuvant chemotherapy.

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.