Kenzo Shimazu

Association of Breast Cancer Stem Cells Identified by Aldehyde Dehydrogenase 1 Expression with Resistance to Sequential Paclitaxel and Epirubicin-Based Chemotherapy for Breast Cancers

Purpose: Breast cancer stem cells have been shown to be associated with resistance to chemotherapy in vitro, but their clinical significance remains to be clarified. The aim of this study was to investigate whether cancer stem cells were clinically significant for resistance to chemotherapy in human breast cancers. Experimental Design: Primary breast cancer patients (n = 108) treated with neoadjuvant chemotherapy consisting of sequential paclitaxel and epirubicin-based chemotherapy were included in the study. Breast cancer stem cells were identified by immunohistochemical staining of CD44/CD24 and aldehyde dehydrogenase 1 (ALDH1) in tumor tissues obtained before and after neoadjuvant chemotherapy. CD44+/CD24− tumor cells or ALDH1-positive tumor cells were considered stem cells. Results: Thirty (27.8%) patients achieved pathologic complete response (pCR). ALDH1-positive tumors were significantly associated with a low pCR rate (9.5% versus 32.2%; P = 0.037), but there was no significant association between CD44+/CD24− tumor cell proportions and pCR rates. Changes in the proportion of CD44+/CD24− or ALDH1-positive tumor cells before and after neoadjuvant chemotherapy were studied in 78 patients who did not achieve pCR. The proportion of ALDH1-positive tumor cells increased significantly (P < 0.001) after neoadjuvant chemotherapy, but that of CD44+/CD24− tumor cells did not. Conclusions: Our findings suggest that breast cancer stem cells identified as ALDH1-positive, but not CD44+/CD24−, play a significant role in resistance to chemotherapy. ALDH1-positive thus seems to be a more significantly predictive marker than CD44+/CD24− for the identification of breast cancer stem cells in terms of resistance to chemotherapy.

Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression.

Recently, aldehyde dehydrogenase (ALDH) 1 has been identified as a reliable marker for breast cancer stem cells. The aim of our study was to investigate the clinicopathological characteristics of breast cancers with ALDH1+ cancer stem cells. In addition, the distribution of ALDH1+ tumor cells was compared on a cell‐by‐cell basis with that of estrogen receptor (ER)+, Ki67+, or human epidermal growth factor receptor type 2 (HER2)+ tumor cells by means of double immunohistochemical staining. Immunohistochemical staining of ALDH1 was applied to 203 primary breast cancers, and the results were compared with various clinicopathological characteristics of breast cancers including tumor size, histological grade, lymph node metastases, lymphovascular invasion, ER, progesterone receptor, HER2, Ki67, and topoisomerase 2A as well as prognosis. Immunohistochemical double staining of ALDH1 and ER, Ki67, or HER2 was also carried out to investigate their distribution. Of the 203 breast cancers, 21 (10%) were found to be ALDH1+, and these cancers were significantly more likely to be ER− (P = 0.004), progesterone receptor− (P = 0.025), HER2+ (P = 0.001), Ki67+ (P < 0.001), and topoisomerase 2A+ tumors (P = 0.012). Immunohistochemical double staining studies showed that ALDH1+ tumor cells were more likely to be ER−, Ki67−, and HER2+ tumor cells. Patients with ALDH1 (score 3+) tumors showed a tendency (P = 0.056) toward a worse prognosis than did those with ALDH1− tumors. Breast cancers with ALDH1+ cancer stem cells posses biologically aggressive phenotypes that tend to have a poor prognosis, and ALDH1+ cancer stem cells are characterized by ER−, Ki67−, and HER2+. (Cancer Sci 2009; 100: 1062–1068)

Endoscopic thyroid surgery through the axillo-bilateral-breast approach.

&NA; We developed a new endoscopic thyroid surgery by the axillo‐bilateralbreast approach (ABBA) method, which is different from the previously described breast approach (BA) in that the port sites are modified to obtain a better view and to prevent the interference of surgical instruments. This modification also improves cosmetic results by eliminating the parasternal incision, which results in hypertrophic scar in a significant number of cases treated with BA. Twelve patients with benign thyroid tumors successfully underwent endoscopic thyroid surgery by ABBA, and their clinical outcomes were compared with those of four patients treated with BA. The mean operation time was significantly shorter in the ABBA group than in the BA group (188 minutes vs. 270 minutes; P < 0.01). Furthermore, the mean blood loss in the ABBA group (53 mL) was half of that in the BA group (108 mL). Neither conversion to open surgery nor significant intraoperative complications were experienced. The operative scars by ABBA became inconspicuous in a few weeks. These results seem to indicate that ABBA is a better method than BA and can be a feasible option, particularly for young patients who opt for the better cosmetic outcome.

Lymphoscintigraphic visualization of internal mammary nodes with subtumoral injection of radiocolloid in patients with breast cancer.

ObjectiveTo determine whether subtumoral injection of radiocolloid is useful for lymphoscintigraphic visualization of the internal mammary node and in sentinel lymph node (SLN) biopsy of the axilla in breast cancer patients. Summary Background DataThe presence of retromammary lymphatics connecting to the axillary and internal mammary basins has been demonstrated by early anatomic studies. Thus, it is hypothesized that some lymph, especially that from the parenchyma under the tumor, may drain into both the axillary and internal mammary basins. MethodsPatients (n = 196) with T1-2, N0 breast cancer underwent preoperative lymphoscintigraphy with radiocolloid (technetium 99m tin colloid) injection into various sites of the breast, followed by SLN biopsy using the combined method with blue dye. Patients were divided into four groups: group A (n = 41), peritumoral injection of both radiocolloid and blue dye; group B (n = 70), periareolar radiocolloid and peritumoral blue dye; group C (n = 45), intradermal radiocolloid and periareolar blue dye; and group D (n = 40), subtumoral radiocolloid and intradermal blue dye. A retrospective analysis of 1,297 breast cancer patients who underwent extended radical mastectomy with internal mammary node dissection was also conducted to determine the relationship between vertical tumor location (superficial or deep) and frequency of axillary and internal mammary node metastases. ResultsOne patient (2%) in group A, 3 (4%) in group B, 0 (0%) in group C, and 15 (38%) in group D exhibited hot spots in the internal mammary region on lymphoscintigraphy (P < .001, group D vs. the other groups). The concordance rate of radiocolloid and blue dye methods in detection of SLNs in the axillary basin was significantly lower in group D than in the other groups. In contrast, the mismatch rate (some SLNs were identified by radiocolloid and other SLNs were identified by blue dye, but no SLN was identified by both in the same patient) was significantly higher in group D than in the other groups. In patients treated with extended radical mastectomy, positivity of axillary and internal mammary metastases was significantly higher in patients (n = 215) with deep tumors than those (n = 368) with superficial tumors. ConclusionsThese results suggest the presence of a retromammary lymphatic pathway from the deep portion of the breast to both axillary and internal mammary basins, which is distinct from the superficial pathway. Therefore, SLN biopsy with a combination of subtumoral and other (peritumoral, dermal, or areolar) injections of radiocolloid will improve both axillary and internal mammary nodal staging.

Sentinel lymph node biopsy using periareolar injection of radiocolloid for patients with neoadjuvant chemotherapy-treated breast carcinoma.

The feasibility and accuracy of sentinel lymph node (SLN) biopsy after neoadjuvant chemotherapy (NAC) for patients with breast carcinoma have been investigated primarily for the situation in which the radiocolloid imaging agent is injected peritumorally. No such study has involved periareolar injection of radiocolloid, although the usefulness of this injection technique has been demonstrated in patients with early‐stage breast carcinoma who have not been treated with NAC. The objective of the current study was to determine the feasibility and accuracy of SLN biopsy using periareolar injection of radiocolloid for patients with breast carcinoma who were treated with NAC.

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)

Prognostic significance of Ki67 index after neoadjuvant chemotherapy in breast cancer

PURPOSE Recently, Ki67 index (cell proliferation marker) has been attracting a considerable attention as a prognostic factor in breast cancer but the prognostic significance of Ki67 after neoadjuvant chemotherapy (NAC) has rarely been examined. EXPERIMENTAL DESIGN Primary breast cancer patients (n = 102) treated with NAC (sequential paclitaxel 12 cycles (q1w) and 5-FU/epirubicin/cyclophosphamide 4 cycles (q3w)) were recruited in the study. Ki67, estrogen receptor (ER) and progesterone receptor (PR) and breast cancer resistant protein (BCRP) and P-glycoprotein were determined by immunohistochemistry and HER2 was determined by FISH in tumor tissues obtained before and after NAC, and their association with patient prognosis (relapse-free survival) was examined. RESULTS Of the 102 patients, pCR was achieved in 30 (29.4%). In the 72 non-pCR patients, Ki67 index significantly (P < 0.001) decreased after NAC. Ki67 index after NAC, but not Ki67 index before NAC, was significantly associated with a patient prognosis (P = 0.022). Multivariate analysis has shown that Ki67 index after NAC is a marginally significant (P = 0.05) prognostic factor and that other biomarkers including ER, PR, BCRP, and P-glycoprotein before and after NAC are not significant. CONCLUSIONS Ki67 after NAC, but not before NAC, is prognostic in breast cancer patients, and might be clinically useful in the prognosis prediction of patients who do not achieve pCR after NAC. On the other hand, BCRP and P-glycoprotein before and after NAC are unlikely to be useful as prognostic factors in these patients.

Indication for sentinel lymph node biopsy for breast cancer when core biopsy shows ductal carcinoma in situ

BACKGROUND The use of sentinel lymph node biopsy (SLNB) for ductal carcinoma in situ (DCIS) is controversial. METHODS A total of 103 primary breast cancer patients who were diagnosed with DCIS by needle biopsy preoperatively and underwent initial SLNB were analyzed retrospectively. RESULTS No sentinel nodal metastasis was detected in 66 patients with the final diagnosis of DCIS. However, 2 (5.4%) of 37 patients with invasive ductal carcinoma at final diagnosis had positive sentinel nodes. Multivariate logistic regression analysis identified 2 independent significant predictors of existence of invasive components: presence of a palpable tumor (odds ratio, 4.091; 95% confidential interval, 1.399-11.959; P = .010) and tumor size of 2.0 cm or larger on magnetic resonance imaging (odds ratio, 4.506; 95% confidence interval, 1.322-15.358; P = .016). CONCLUSIONS Initial SLNB should be considered for patients diagnosed with DCIS by needle biopsy when they have a high risk for harboring invasive ductal cancer preoperatively.

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.

Methylated DNA and Total DNA in Serum Detected by One-Step Methylation-Specific PCR Is Predictive of Poor Prognosis for Breast Cancer Patients

Purpose: We recently developed the one-step methylation-specific PCR (OS-MSP) assay which can detect methylated DNA (met-DNA) in serum with high sensitivity. To examine its prognostic value, we applied this new assay to the detection of met-DNA in serum of breast cancer patients. Methods: Serum samples taken before surgery from 336 primary invasive breast cancer patients were subjected to the OS-MSP assay for the promoter regions of GSTP1, RASSF1A, and RARβ2. The assay outcome was considered positive when methylation was detected in at least one of these three genes. Total DNA content in serum was also determined. Results: Of the 336 stage I/II patients, 33 (10%) were positive for met-DNA in serum and showed a significantly worse overall survival (OS) rate at 100 months (78 vs. 95%; p = 0.002) than those with negative findings (n = 303). Patients with high total DNA in serum (n = 112) also showed a significantly worse OS rate at 100 months (86 vs. 97%; p = 0.001) than those with low total DNA in serum (n = 224). Moreover, patients both positive for met-DNA and with high total DNA in serum (n = 18) showed a much worse OS rate at 100 months (65 vs. 94%; p < 0.001) than the others (n = 318). Conclusions: Met-DNA in serum detected with the OS-MSP assay constitutes a significant and independent prognostic factor, and its combination with total DNA in serum seems to be even more effective for prediction of prognosis for breast cancer patients.