Utility of intraoperative frozen section examinations of surgical margins: implication of margin-exposed tumor component features on further surgical treatment.

OBJECTIVE: In patients who underwent breast-conserving surgery, we attempted to identify the histological characteristics of margin-exposed tumor components on intraoperative frozen section examinations that were predictive of residual tumor components in additionally resected specimens. METHODS: Of 1835 patients who underwent breast-conserving surgery, we identified 220 patients who had positive surgical margins determined by intraoperative frozen section examinations and who had undergone immediate additional resections. Two observers (M.K., H.T.) reviewed the slides of frozen sections and confirmed the presence of tumor components. RESULTS: In additionally resected specimens, residual tumors were detected in 115 cases (52.3%) but not in 105 cases (47.7%). The primary tumor characteristics of extensive intraductal component (+), younger age, invasive lobular carcinoma and pathological T3 classification were significantly associated with the residual tumor components. The margin-exposed tumor components of the maximum diameter, number of positive margins and histological type were correlated with the residual tumors. Multivariate analysis showed that the maximum tumor diameter was an independent risk factor for residual tumors. CONCLUSIONS: Diagnosis of positive margins by intraoperative frozen section examinations was useful for predicting residual tumors, and three histological properties of the margin-exposed tumor components were correlated with the status of residual tumor components. Although it was impossible to clearly identify the single main factor for predicting patients for whom additional resections were not necessary, it may be possible to consider stratification of additional surgical therapy according to the characteristics of margin-exposed tumor components on intraoperative frozen section examinations.

[Application of epigenetics to cancer diagnosis and therapy].

Epigenetic modifications are responsible for the stable maintenance of cellular phenotypes and consist of DNA methylation and histone modifications. Epigenetic abnormalities can be causally involved in cancer development (driver) and can also be present as a passenger. Aberrant DNA methylation has unique characteristics different from point mutations and is utilized as a diagnostic target. First, aberrant DNA methylation can be present at a high level in noncancerous tissues, and because its level can correlate with cancer risk, it is useful as a cancer risk marker. Second, aberrant DNA methylation can be detected with high sensitivity and therefore it is used to detect cancer cells. Third, DNA methylation is stable at different time points, such as at biopsy and during chemotherapy, and provides a good source of biomarkers. In addition, resistance to contaminating cells makes DNA methylation useful in genome-wide screening of biomarkers for various clinicopathologic characteristics. From the therapeutic viewpoint, DNA demethylating agents have been approved for the treatment of myelodysplastic syndrome and histone deacetylase inhibitors for the treatment of cutaneous T-cell lymphoma. The next steps include increasing the specificity for target genes, the development of biomarkers for dose and response studies, and establishing a strategy to select drugs for combination therapy. The further use of epigenetics for diagnosis and therapy is warranted.

Development of a novel approach, the epigenome-based outlier approach, to identify tumor-suppressor genes silenced by aberrant DNA methylation.

Identification of tumor-suppressor genes (TSGs) silenced by aberrant methylation of promoter CpG islands (CGIs) is important, but hampered by a large number of genes methylated as passengers of carcinogenesis. To overcome this issue, we here took advantage of the fact that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. First, we demonstrated that three of six known TSGs in breast cancer and two of three in colon cancer had Pol II and lacked H3K27me3 in normal cells, being outliers to the general rule. BRCA1, HOXA5, MLH1, and RASSF1A had high Pol II, but were expressed only at low levels in normal cells, and were unlikely to be identified as outliers by their expression statuses in normal cells. Then, using epigenome statuses (Pol II binding and H3K27me3) in normal cells, we made a genome-wide search for outliers in breast cancers, and identified 14 outlier promoter CGIs. Among these, DZIP1, FBN2, HOXA5, and HOXC9 were confirmed to be methylated in primary breast cancer samples. Knockdown of DZIP1 in breast cancer cell lines led to increases of their growth, suggesting it to be a novel TSG. The outliers based on their epigenome statuses contained unique TSGs, including DZIP1, compared with those identified by the expression microarray data. These results showed that the epigenome-based outlier approach is capable of identifying a different set of TSGs, compared to the expression-based outlier approach.

Methylation silencing of angiopoietin-like 4 in rat and human mammary carcinomas.

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2'-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced.

Evaluation of sentinel node biopsy by combined fluorescent and dye method and lymph flow for breast cancer.

BACKGROUND: Conservative breast resection with subsequent sentinel lymph node biopsy (SNB) is an increasingly popular initial approach for the treatment of breast cancer due to decreased invasiveness. SNB is a shorter procedure with fewer side effects than more substantial surgical procedures, but it sometimes fails to identify metastatic disease. Therefore, a highly sensitive and convenient method is needed to identify sentinel lymph nodes (SLN) with a high probability of containing disease in SNB. We compared the combination of radioisotope or dye with a fluorescence compound to analyze lymph flow to identify targets for SNB. MATERIALS AND METHODS: We examined patients with breast cancer lacking metastases in the axillary lymph node (ALN). Two methods for targeted SNB were developed: (1) Indocyanine Green (ICG) and Patent blue were injected into the skin overlying the tumor and sub-areolar region just before the surgical procedure. (2) ICG and radiocolloid were injected into the skin overlying the tumor and sub-areolar region. The draining fluorescent lymphatic duct was visualized using a Photodynamic Eye (PDE). We removed the SLNs that were identified by the dye and fluorescence imaging methods. Method 1 was applied to 113 patients undergoing SNB, and 29 patients were treated with Method 2. In our study, patients were grouped by lymph flow into two types: Type C demonstrated convergence to one lymph duct. Type S demonstrated separate lymph ducts. RESULTS: Using the fluorescence imaging method, 99.3% of SLNs were identified, and 3.8 SLNs per patient were seen. The SLN identification rates for Patent blue dye and radiocolloid were 92.9% and 100%, respectively, while 1.9 and 2.0 SLNs per patient, respectively, were seen with these methods. We classified two types of lymph flow based on the pattern of lymphatic drainage. Type C converged to a single lymph duct, while Type S drained to separate ducts. Type S lymph drainage was seen in 29/142 patients (20.4%), and Type C drainage was found in 113/141 patients (79.6%). Of the patients with Type S drainage, there were 4.1 SLNs per patient, but only 3.4 SLNs per patient were seen in individuals with Type C drainage. Forty cases had metastases found in the ALNs, and five of these cases were dye-negative and fluorescence-positive. Among these cases, the average number of SLNs identified was one. CONCLUSION: The combination of fluorescence with a visible dye is a highly sensitive method for SLN identification. When SNB is guided by only the dye method, there is a risk of missing appropriate SLNs in patients with Type S lymph drainage or weak dye staining. The use of a fluorescence method together with dye could increase sensitivity of detection in these cases. Furthermore, fluorescent methods are ideal for hospitals that cannot use conventional radioactive measures.