Evaluation of 2014 margin guidelines on re-excision and recurrence rates after breast conserving surgery: A multi-institution retrospective study.

INTRODUCTION: A 2014 consensus statement from the Society of Surgical Oncology and American Society for Radiation Oncology supported "no ink on tumor" as an adequate margin for breast conserving therapy (BCT). This study evaluates this statement in a multi-institution cohort. METHODS: A retrospective review of BCT cases at 3 comprehensive cancer centers was performed. Women age >18 receiving BCT for T1-2 breast cancer from 2008-2012 were included. Pre-2014, all sites considered 2 mm adequate. Estimated re-excision rates using the 2014 guidelines were calculated and factors predictive of re-excision were analyzed. RESULTS: 542 patients (545 lumpectomies) were eligible. Using a ≥2 mm margin standard, 32.8% of patients underwent re-excision compared to 14.1% after 2014 (p < 0.0001). Tumor size (p= 0.003), grade (p=0.015), and lymphovascular invasion (p=0.021) were predictive of re-excision. Patients with additional intraoperative margins excised were less likely to require reoperation (p=0.002). Local recurrence was unaffected by re-excision after mean followup of 66 months. CONCLUSIONS: The 2014 margin guidelines markedly reduce re-excision rates. There is no difference in local recurrence for patients after re-excision for a close margin versus without Powered.

Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii.

BACKGROUND: Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. METHODS: We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. RESULTS: The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species.

Comparative transcriptome among Euscaphis konishii Hayata tissues and analysis of genes involved in flavonoid biosynthesis and accumulation.

BACHGROUND: Euscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform. RESULTS: A total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60 bp and N50 length of 1307 nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids. CONCLUSION: We present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

The complete chloroplast genome sequence of Euscaphis japonica (Staphyleaceae).

Euscaphis japonica is not only an ideal ornamental plant, but also a traditional medicinal plant, which is an extremely valuable species to study. We determined the complete chloroplast genome sequence for E. japonica using Illumina sequencing technology. The complete chloroplast sequence is 160,467 bp in length, including a large single-copy (LSC) region of 88,716 bp, a small single-copy (SSC) region of 18,614 bp, and a pair of invert repeats (IR) regions of 26,568 bp. Plastid genome contains 142 genes, 46 protein-coding genes, 39 tRNA genes, and eight rRNA genes. Phylogenetic analysis base on 14 chloroplast genomes indicates that E. japonica forms an isolated clade and sisters to Glycosmis-Gossypium clade with strong support.

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data.

BACKGROUND: Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. RESULTS: In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets. CONCLUSION: Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.

Use of axillary lymph node dissection (ALND) in patients with micrometastatic breast cancer.

BACKGROUND: Sentinel lymph node (SLN) biopsy is the current prognostic tool for clinically node-negative breast cancer patients. If the SLN reveals macrometastasis, axillary lymph node dissection (ALND) is recommended. However, the use of ALND in patients with micrometastasis is debated. The objective of this study was to assess the utilization of ALND in the treatment of micrometastatic breast cancer. METHODS: An IRB approved, retrospective study of a pooled dataset of breast cancer patients with micrometastatic disease on SLN biopsy was performed. Patients diagnosed from 1999-2016 were identified via query of a single-institution National Comprehensive Cancer Network (NCCN) breast cancer database as well as a prospective tumor board. RESULTS: A total of 91 patients were diagnosed with micrometastatic nodal disease. The median age at diagnosis was 56 y (range: 31-85); median follow-up time was 47 mo (range: 0-203 mo). 42/91(46.2%) patients had ALND of which 37/42 (88.1%) were a second operation; 3/42(7.1%) patients had additional positive nodes found at ALND. 44/91 (48.4%) patients received radiation. 7/91 (7.7%) patients had a recurrence, 5/7 local, including one axillary (2.1%; patient declined ALND). CONCLUSIONS: Given that the risk of lymphedema after ALND ranges between 20%-53%, the morbidity of ALND may far exceed the likelihood of detecting further nodal involvement in women with micrometastatic disease: 7.1% in this series.

Menopausal status does not predict Oncotype DX recurrence score.

BACKGROUND: Adjuvant treatment for early stage, estrogen receptor (ER) positive invasive breast cancer has been based on prognosticators such as menopausal status. The recurrence score (RS) from the 21-gene assay Oncotype DX (ODX) is predictive of a 10-y distant recurrence in this population but is rarely applied to premenopausal patients. The relationship between menopausal status and RS was evaluated. MATERIALS AND METHODS: An institutional review board-approved retrospective review was conducted of invasive breast cancer patients with known RS. ODX eligibility was based on National Comprehensive Cancer Network guidelines or physician discretion. Perimenopausal women were classified as premenopausal for statistical analyses. Comparisons of menopausal status and RS were made using general linear regression model and the exact Wilcoxon rank-sum test. RESULTS: Menopausal status was available for 575 patients (142 premenopausal, 433 postmenopausal). Median age was 46 y for premenopausal and 62 y for postmenopausal. Median invasive tumor size was 1.5 cm for both cohorts. Mastectomy rate was higher in the premenopausal group (54.8%) than postmenopausal (42%; P = 0.0001). Premenopausal women had a higher local-regional recurrence rate (2.8% versus 0%; P = 0.0384) but distant recurrence and overall survival were not statistically different (P = 0.6808). Median ER H-score was lower in premenopausal (H-score = 270) than postmenopausal women (H-score = 280; P < 0.0001). Median RS was 16 for both premenopausal (range, 0-54) and postmenopausal (range, 0-63) women. Menopausal status as a categorical variable was not predictive of RS (P-value = 0.6780). CONCLUSIONS: Menopausal status has limited predictive power for distant recurrence. Therefore, menopausal status alone should not preclude performance of ODX in ER-positive, early stage breast cancer.

Ipsilateral nodal recurrence after axillary dissection for breast cancer.

INTRODUCTION: Level I/II axillary lymph node dissection (ALND) is the standard operation for patients with node-positive breast cancer. The objective of this study was to assess the incidence of regional nodal recurrence (RNR) after ALND performed for definitive operative treatment for primary breast cancer. MATERIALS AND METHODS: A retrospective, Institutional Review Board-approved query of our single-institution National Comprehensive Cancer Network database was performed for patients undergoing ALND who developed subsequent RNR. All patients were treated from 1999 to 2009. A detailed chart review was performed and clinical, pathologic, treatment, and outcome data were collected. RESULTS: A total of 1614 patients had an ALND for initial staging; 14/1614 (0.9%) patients had RNR. Two other patients had contralateral breast/axillary recurrences and were excluded. The mean age at diagnosis for the sample group was 52.7 y (range 34-77); mean follow-up time was 47.1 mo (range 12.6-114.6). The median number of nodes for ALND was 16 (range 8-27). The median number of positive nodes was 2.5 (range 0-7). Nine (64.3%) cases were estrogen receptor/progesterone receptor negative. Twelve (85.7%) patients had axillary recurrences, and six of 12 (50.0%) had concurrent chest wall lesions. Twelve patients (85.7%) had distant metastases; nine of 12 (75.0%) died; two were lost to follow-up. Mean time from RNR to distant recurrence was 6.0 mo (range 0-29.3 mo). CONCLUSIONS: RNR after ALND is rare but a harbinger of poor outcome. This is apparent regardless of treatment used for initial disease or recurrence. Specifically, RNR after primary ALND is related to increased risk of mortality and distant metastatic disease.