Masahiko Tsujimoto

One-step Nucleic Acid Amplification for Intraoperative Detection of Lymph Node Metastasis in Breast Cancer Patients

Purpose: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. Experimental Design: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry–based three-level histopathologic examination. The results from the two methods were then compared. Results: We established CK19 mRNA cutoff values of 2.5 × 102 and 5 × 103 copies/μL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. Conclusion: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.

Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma

In order to elucidate the clinical significance of the erbB family, epidermal growth factor receptor (EGF-R), c-erbB-2, c-erbB-3 and c-erbB-4 in hepatocellular carcinoma (HCC), we investigated the expression of these proteins by means of immunohistochemistry for HCC as well as adjacent noncancerous lesions. EGF-R was expressed in 68% of the HCC examined and showed correlation with the proliferating activity, stage, intrahepatic metastasis and carcinoma differentiation. c-erbB-2 was expressed in only 21% of the cases and showed no relationships with the clinicopathological parameters. c-erbB-3 protein was observed in 84% of the HCC and 38.1% of the noncancerous lesions. Its expression in HCC was equal to or greater than noncancerous lesions in 90.5% of the cases, and was related to the stage, portal invasion, cell proliferating activity, tumour size, intrahepatic metastasis and carcinoma differentiation. c-erbB-4 protein was expressed in 61.0% of HCC and in as much as 86.1% of the noncancerous lesions. Unlike the expression of c-erbB-3, that of c-erbB-4 in HCC was less than that of the adjacent noncancerous lesions in 51.2% of the cases. No statistical significance could be established between this protein expression in HCC and clinicopathological features. EGF-R and c-erbB-3 affected disease-free survival, but were not recognized as independent prognostic factors by multivariate analysis. The present study suggests that, of the four receptors, EGF-R and c-erbB-3 play important roles in the progression of HCC.

Prognostic significance of Ki‐67 reactivity in soft tissue sarcomas

Proliferative activity of soft tissue sarcomas (STS) in 34 cases was estimated by immunohistochemical procedures (avidin‐biotin complex [ABC] method) with monoclonal antibody Ki‐67 which reacts with a nuclear antigen expressed in all phases of cell cycle except G0. In 20 of 34 cases (59%), varying numbers of Ki‐67‐positive tumor cells were detected with a range from 5 to 382 per 10 high power fields (HPF) (mean 57.2/10 HPF). Ki‐67 index (the number of Ki‐67‐positive tumor cells/10 HPF) positively correlated with mitotic count (r = 0.428, P < 0.02), cellularity (r = 0.447, P < 0.01), and histologic grade (r = 0.473, P < 0.01). The Ki‐67 low index group (less than 50/10 HPF) showed more favorable prognosis than the high index group (more than 50/10 HPF) (P < 0.005). Three cases with low mitotic count and unfavorable prognosis were proved to be the Ki‐67 high index group (142‐382/10 HPF). These results indicated that reactivity of tumor cells for Ki‐67 is a useful prognostic marker in the patients with STS, and might be used as one of the histologic factors for the grading of STS.

Molecular Detection of Lymph Node Metastases in Breast Cancer Patients: Results of a Multicenter Trial Using the One-Step Nucleic Acid Amplification Assay

Purpose: Accurate assessment of metastasis in sentinel lymph nodes (SLN) of breast cancer is important but involves a heavy workload for the pathologist. We conducted a multicenter clinical trial in Japan to evaluate a new automated assay system for cytokeratin 19 mRNA, the one-step nucleic acid amplification (OSNA) assay (Sysmex), to detect lymph node metastasis of breast cancer. Experimental Design: Surgically obtained axillary lymph nodes were sectioned into four pieces, two of which were examined with the OSNA assay. The other two adjacent pieces were examined with H&E and immunohistochemical staining for cytokeratin 19. Serial sections at 0.2-mm intervals were used in trial 1 to determine the specificity of the OSNA assay, and three pairs of sections cut from the sliced surfaces of the pieces were used in trial 2 to compare the accuracy of the OSNA assay with that of a routine pathologic examination for SLNs in Japan. Results: In trial 1, the sensitivity and specificity were 95.0% [95% confidence interval (95% CI), 75.1-99.9%] and 97.1% (95% CI, 91.8-99.4%), respectively, for 124 axillary lymph nodes obtained from 34 patients. In trial 2, the agreement between findings of the assay and of the pathologic examination was 92.9% (95% CI, 90.1-95.1%) for 450 axillary lymph nodes obtained from 164 patients. Conclusion: The OSNA assay can detect lymph node metastasis as accurately as can conventional pathology and thus can be an effective addition to or alternative for rapid intraoperative examination of SLNs.

Lymph vessel density correlates with nodal status, VEGF-C expression, and prognosis in breast cancer.

Metastasis to the regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer and an important prognostic factor in many types of cancer. Recent evidence suggests that VEGF-C promotes lymphangiogenesis, and that tumor lymphangiogenesis in turn promotes lymphatic metastasis. We have studied the role of LVD in breast cancer, and examined whether LVD is associated with lymph node metastasis, VEGF-C expression, or prognosis. In addition, we examined whether VEGF-C mRNA transcript levels were associated with lymph node metastasis and LVD. We began by investigating the lymphatics in primary human breast carcinoma with long-term follow-up (113 cases of invasive ductal and other breast cancers) by quantitative immunohistochemical staining for podoplanin. We then analyzed the relationship between LVD and lymph node status as well as VEGF-C immunoreactivity and other established clinicopathological parameters. The relationship between LVD and prognosis was also studied. VEGF-C mRNA transcript levels were examined by quantitative real-time RT-PCR, in 55 invasive ductal breast carcinomas. This was followed by an analysis of the relationship between VEGF-C mRNA transcript levels and lymph node metastasis as well as LVD. Mean LVD of ‘hot spots’ was 10.2 ± 7.4/each case. LVD was significantly correlated with lymph node metastasis (p < 0.0001), VEGF-C immunoreactivity (p = 0.0084), and podoplanin positive lymphatic invasion (p < 0.0001). Survival curves determined by the Kaplan–Meier method and univariate analysis demonstrated that high LVD was associated with both worse disease free survival (p = 0.0033) and overall survival (p = 0.0391). VEGF-C mRNA transcript levels were also correlated with lymph node metastasis (p = 0.0074) and LVD (p = 0.0409). Increased LVD was correlated with lymph node metastasis and VEGF-C expression. High LVD may be a significant unfavorable prognostic factor for long-term survival in breast cancer.

mTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovary.

Purpose: Mammalian target of rapamycin (mTOR) plays a central role in cell proliferation and is regarded as a promising target in cancer therapy, including for ovarian cancer. This study aimed to examine the role of mTOR as a therapeutic target in clear cell carcinoma of the ovary, which is regarded as an aggressive, chemoresistant histologic subtype. Experimental Design: Using tissue microarrays of 98 primary ovarian cancers (52 clear cell carcinomas and 46 serous adenocarcinomas), the expression of phospho-mTOR was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTOR inhibition by RAD001 (everolimus) was examined using two pairs of cisplatin-sensitive parental (RMG1 and KOC7C) and cisplatin-resistant human clear cell carcinoma cell lines (RMG1-CR and KOC7C-CR) both in vitro and in vivo. Results: Immunohistochemical analysis showed that mTOR was more frequently activated in clear cell carcinomas than in serous adenocarcinomas (86.6% versus 50%). Treatment with RAD001 markedly inhibited the growth of both RMG1 and KOC7C cells both in vitro and in vivo. Increased expression of phospho-mTOR was observed in cisplatin-resistant RMG1-CR and KOC7C-CR cells, compared with the respective parental cells. This increased expression of phospho-mTOR in cisplatin-resistant cells was associated with increased activation of AKT. RMG1-CR and KOC7C-CR cells showed greater sensitivity to RAD001 than did parental RMG1 and KOC7C cells, respectively, in vitro and in vivo. Conclusion: mTOR is frequently activated in clear cell carcinoma and can be a promising therapeutic target in the management of clear cell carcinoma. Moreover, mTOR inhibition by RAD001 may be efficacious as a second-line treatment of recurrent disease in patients previously treated with cisplatin. (Clin Cancer Res 2009;15(17):5404–13)

Clinicopathological Significance of Poorly Differentiated Thyroid Carcinoma

The clinicopathological importance of a heterogeneous group of poorly differentiated thyroid carcinomas is not fully understood. Using data obtained from 303 surgically treated patients with differentiated thyroid carcinomas, the correlations between the aggressive histologic features and the clinicopathological findings, postoperative recurrences, and prognosis were retrospectively examined. In 201 cases, the carcinomas were well differentiated. The remaining 102 cases of poorly differentiated carcinomas were divided into two groups: focal-poorly differentiated (<10%) and diffuse-poorly differentiated (> or = 10%) carcinomas according to the extent of the poorly differentiated component. These poorly differentiated carcinomas were associated with high age (focal-poorly differentiated and diffuse-poorly differentiated versus well differentiated: 55 years and 59 versus 49; p < 0.0001), frequent presence of lymph node metastases (70% and 66% versus 48%; p = 0.0099) and distant metastases at diagnosis (11% and 11% versus 2%; p = 0.0098), and extrathyroidal invasion (53% and 53% versus 21%; p < 0.0001). There was independent correlation with age and the presence of extrathyroidal invasion. Cases of diffuse-poorly differentiated carcinomas showed frequent relapse (diffuse-poorly differentiated versus focal-poorly differentiated and well differentiated: 45% versus 30% and 24%; p = 0.0062) and poor prognoses (mean survival period = 9.15 versus 19.03 and 20.87 years; p < 0.0001) compared with the well and focal-poorly differentiated carcinomas. These data suggest that diffuse-poorly differentiated carcinoma is an important clinicopathological entity.

Multivariate analysis for histologic prognostic factors in soft tissue sarcomas

To investigate histologic prognostic factors in soft tissue sarcomas (STS), multivariate analysis was performed on 236 patients with complete clinical information. These included 141 males and 95 females with an age range from 1 to 85 years (median, 47.6 years). Histologically, malignant fibrous histiocytoma (33.5%) was the most common type followed by synovial sarcoma (11%), liposarcoma (10%), rhabdomyosarcoma (7%), and leiomyosarcoma (7%). Monofactorial analysis revealed that sex, depth, location, histologic grade, cellularity, frequency of mitosis, and necrosis were significant prognostic factors. By multivariate analysis, only frequency of mitosis and depth of tumors were proved to be of prognostic significance. The prognostic importance of the frequency of mitosis as shown in previous and the present studies indicates that further investigation of the cell proliferation in patients with STS is required.

Non-Hodgkin's lymphomas in Osaka, Japan

Six hundred and eight-four cases with non-Hodgkins lymphomas (NHL) in Osaka, Japan were reviewed, using current histopathological classification to obtain a general feature of Japanese lymphomas. There were no remarkable differences in the distribution of age, sex and the location of primary tumors between Japanese NHL and the disease in Western races. Histologically, however, several differences were present; in particular, the lower frequencies of nodular lymphomas of nodal origin and lymphocytic type of nodal and extranodal origin in the present cases. About 10% of the cases show a peculiar histology characteristic for adult T cell lymphomas. From this study, it appears that the ratio of low-grade malignant NHL in Japan is much lower than in the Western countries.

Distal Protection Improved Reperfusion and Reduced Left Ventricular Dysfunction in Patients With Acute Myocardial Infarction Who Had Angioscopically Defined Ruptured Plaque

Background—Distal protection, in the Saphenous Vein Graft Angioplasty Free of Emboli (SAFER) trial, is demonstrated to prevent distal embolism in the percutaneous coronary intervention of saphenous vein graft. However, in the Enhanced Myocardial Efficacy and Recovery by Aspiration of Liberated Debris (EMERALD) trial, it was not effective in the percutaneous coronary intervention of native coronary arteries in patients with acute myocardial infarction (AMI). We hypothesized that its effectiveness would be determined by lesion characteristics. Therefore, we classified the type of culprit lesion by angioscopy and examined its influence on the effectiveness of distal protection, comparing patients with AMI treated with and without distal protection. Methods and Results—Consecutive patients with AMI treated without distal protection (n=110) from July 2000 to July 2002 and those treated with distal protection (n=81) from July 2002 to July 2004 were included. Patients in each group were subdivided according to whether or not they had angioscopically defined ruptured plaque at culprit lesion. Among those groups, incidence of no-reflow phenomenon, ST-segment resolution, myocardial blush grade, and left ventricular ejection fraction at 6 months were compared. Aspirated samples by distal protection were semiquantitatively and histologically analyzed and compared between patients with and without ruptured plaque. No-reflow phenomenon was most frequently (P<0.05) observed in patients with ruptured plaque treated without distal protection. ST-segment resolution (68±15% versus 40±21%, P<0.001), myocardial blush grade (2.6±0.5 versus 1.8±0.3, P<0.001), and left ventricular ejection fraction (47.2±6.7% versus 41.0±9.7%, P<0.01) were improved by distal protection among patients with ruptured plaque but not among patients without ruptured plaque. Aspirated samples >1 mm were detected more frequently (97.3% versus 78.5%, P<0.05) in patients with ruptured plaque than those without ruptured plaque. Histologically, aspirated samples contained plaque debris (95.3% versus 31.1%, P<0.05) more frequently in patients with ruptured plaque than in those without ruptured plaque. Conclusions—Distal protection reduced microcirculation damage and left ventricular dysfunction in patients with AMI who had angioscopically defined ruptured plaque. Distal embolization of plaque debris was detected more frequently in patients with ruptured plaque. These results suggest that microcirculation damage and left ventricular dysfunction are increased mainly by distal embolization of plaque debris rather than of thrombus.