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Corymbia (a eucalypt) is an important forestry genus and a potential lignocellulosic bioenergy feedstock. The composition of the lignocellulosic cell wall significantly impacts pretreatment efficiency and conversion to biofuel but is variable and changes with age. In this study, we estimated Klason lignin content, composition, and monosaccharide (glucose and xylose) release after enzymatic saccharification of untreated and hydrothermally pretreated biomass from Corymbia parental species Corymbia torelliana (CT), Corymbia citriodora subsp. variegata (spotted gum; CCV), and interspecific F1 hybrids (CT × CCV) at ages 9 and 12 years from planting. Analysis of lignin composition derived from syringyl/guaiacyl monolignols (S/G) found significant differences among taxa, with CT S/G ratios (2.2 and 2.0) being significantly lower than CCV (2.6 and 2.3) or hybrids (2.5 and 2.3) at ages 9 and 12 respectively. In general, enzymatic saccharification yields from untreated biomass were significantly different among taxa, with CT (113 and 75 mg g−1) and hybrids (108 and 81 mg g−1) yielding significantly higher glucose from untreated biomass than CCV (82 and 56 mg g−1) at ages 9 and 12 respectively. Comparison of traits within taxa between ages 9 and 12 found S/G ratios and glucose yields from untreated biomass were significantly lower in CT, CCV and hybrid taxa. In conclusion, the formation of lignocellulosic cell walls is complex, influenced by genetics and age of material, requiring optimization of rotation age for biofuel production and other industrial processes.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Importance: Adding fulvestrant to anastrozole (A+F) improved survival in postmenopausal women with advanced estrogen receptor (ER)-positive/ERBB2 (formerly HER2)-negative breast cancer. However, the combination has not been tested in early-stage disease., Objective: To determine whether neoadjuvant fulvestrant or A+F increases the rate of pathologic complete response or ypT1-2N0/N1mic/Ki67 2.7% or less residual disease (referred to as endocrine-sensitive disease) over anastrozole alone., Design, Setting, and Participants: A phase 3 randomized clinical trial assessing differences in clinical and correlative outcomes between each of the fulvestrant-containing arms and the anastrozole arm. Postmenopausal women with clinical stage II to III, ER-rich (Allred score 6-8 or >66%)/ERBB2-negative breast cancer were included. All analyses were based on data frozen on March 2, 2023., Interventions: Patients received anastrozole, fulvestrant, or a combination for 6 months preoperatively. Tumor Ki67 was assessed at week 4 and optionally at week 12, and if greater than 10% at either time point, the patient switched to neoadjuvant chemotherapy or immediate surgery., Main Outcomes and Measures: The primary outcome was the endocrine-sensitive disease rate (ESDR). A secondary outcome was the percentage change in Ki67 after 4 weeks of neoadjuvant endocrine therapy (NET) (week 4 Ki67 suppression)., Results: Between February 2014 and November 2018, 1362 female patients (mean [SD] age, 65.0 [8.2] years) were enrolled. Among the 1298 evaluable patients, ESDRs were 18.7% (95% CI, 15.1%-22.7%), 22.8% (95% CI, 18.9%-27.1%), and 20.5% (95% CI, 16.8%-24.6%) with anastrozole, fulvestrant, and A+F, respectively. Compared to anastrozole, neither fulvestrant-containing regimen significantly improved ESDR or week 4 Ki67 suppression. The rate of week 4 or week 12 Ki67 greater than 10% was 25.1%, 24.2%, and 15.7% with anastrozole, fulvestrant, and A+F, respectively. Pathologic complete response/residual cancer burden class I occurred in 8 of 167 patients and 17 of 167 patients, respectively (15.0%; 95% CI, 9.9%-21.3%), after switching to neoadjuvant chemotherapy due to week 4 or week 12 Ki67 greater than 10%. PAM50 subtyping derived from RNA sequencing of baseline biopsies available for 753 patients (58%) identified 394 luminal A, 304 luminal B, and 55 nonluminal tumors. A+F led to a greater week 4 Ki67 suppression than anastrozole alone in luminal B tumors (median [IQR], -90.4% [-95.2 to -81.9%] vs -76.7% [-89.0 to -55.6%]; P < .001), but not luminal A tumors. Thirty-six nonluminal tumors (65.5%) had a week 4 or week 12 Ki67 greater than 10%., Conclusions and Relevance: In this randomized clinical trial, neither fulvestrant nor A+F significantly improved the 6-month ESDR over anastrozole in ER-rich/ERBB2-negative breast cancer. Aromatase inhibition remains the standard-of-care NET. Differential NET response by PAM50 subtype in exploratory analyses warrants further investigation., Trial Registration: ClinicalTrials.gov Identifier: NCT01953588.

Importance: Oral tamoxifen citrate benefits women with ductal carcinoma in situ (DCIS), but concern about toxic effects has limited acceptance. Previous pilot studies have suggested transdermal 4-hydroxytamoxifen gel has equivalent antiproliferative efficacy to oral tamoxifen, with low systemic exposure., Objective: To demonstrate that 4-hydroxytamoxifen gel applied to the breast skin is noninferior to oral tamoxifen in its antiproliferative effect in DCIS lesions., Design, Setting, and Participants: This randomized, double-blind, phase 2 preoperative window trial was performed at multicenter breast surgery referral practices from May 31, 2017, to January 27, 2021. Among 408 women with estrogen receptor-positive DCIS who were approached, 120 consented and 100 initiated study treatment. The most common reasons for nonparticipation were surgical delay, disinterest in research, and concerns about toxic effects. Data were analyzed from January 26, 2021, to October 5, 2022., Intervention: Random assignment to oral tamoxifen citrate, 20 mg/d, and gel placebo or 4-hydroxytamoxifen gel, 2 mg/d per breast, and oral placebo, for 4 to 10 weeks, followed by DCIS resection., Main Outcomes and Measures: The primary end point was absolute change in DCIS Ki-67 labeling index (Ki67-LI). Secondary end points included 12-gene DCIS Score, breast tissue tamoxifen metabolite concentrations, tamoxifen-responsive plasma protein levels, and patient-reported symptoms. Noninferiority of Ki67-LI reduction by 4-hydroxytamoxifen gel was tested using analysis of covariance; within- and between-arm comparisons were performed with paired t tests for mean values or the Wilcoxon rank sum test for medians., Results: Of 90 participants completing treatment (mean [SD] age, 55 [11] years; 8 [8.9%] Asian, 16 [17.8%] Black, 8 [8.9%] Latina, and 53 [58.9%] White), 15 lacked residual DCIS in the surgical sample, leaving 75 evaluable for the primary end point analysis (40 in the oral tamoxifen group and 35 in the 4-hydroxytamoxifen gel group). Posttreatment Ki67-LI was 3.3% higher (80% CI, 2.1%-4.6%) in the 4-hydroxytamoxifen gel group compared with the oral tamoxifen group, exceeding the noninferiority margin (2.6%). The DCIS Score decreased more with oral tamoxifen treatment (-16 [95% CI, -22 to -9.4]) than with 4-hydroxytamoxifen gel (-1.8 [95% CI, -5.8 to 2.3]). The median 4-hydroxytamoxifen concentrations deep in the breast were nonsignificantly higher in the oral tamoxifen group (5.7 [IQR, 4.0-7.9] vs 3.8 [IQR, 1.3-7.9] ng/g), whereas endoxifen was abundant in the oral tamoxifen group and minimal in the 4-hydroxytamoxifen gel group (median, 13.0 [IQR, 8.9-20.6] vs 0.3 [IQR, 0-0.3] ng/g; P < .001). Oral tamoxifen caused expected adverse changes in plasma protein levels and vasomotor symptoms, with minimal changes in the transdermal group., Conclusions and Relevance: In this randomized clinical trial, antiproliferative noninferiority of 4-hydroxytamoxifen gel to oral tamoxifen was not confirmed, potentially owing to endoxifen exposure differences. New transdermal approaches must deliver higher drug quantities and/or include the most potent metabolites., Trial Registration: ClinicalTrials.gov Identifier: NCT02993159.

Background: The 31-gene expression profile test (Class 1A: low-risk; 1B/2A: intermediate-risk; 2B: high-risk) is validated to identify patients with cutaneous melanoma who can safely forego sentinel lymph node biopsy (SLNB). The objective of the current study is to quantify SLNB reduction by clinicians using 31-GEP., Methods: Patients with T1-T2 tumors eligible for SLNB were seen by surgical oncologists (89.1%), dermatologists (7.8%), and medical oncologists (3.1%). After receiving 31-GEP results but before SLNB, clinicians were asked which clinical and pathological features influenced SLNB decisions ( n  = 191). The Exact binomial test was used to compare SLNB procedure rates to a contemporary study (78% SLNB baseline rate). Logistic regression modeling (odds ratio [OR], 95% CI) was used to identify features associated with SLNB procedure rates., Results: One hundred clinical decisions (52.4%) were influenced by the 31-GEP to forego SLNB and 70% (70/100) were not performed. Of the 30 performed, 0% (0/30) were positive. The 31-GEP influenced sixty-three clinical decisions (33.0%) to perform SLNB, and 92.1% (58/63) were performed. There was a clinically meaningful 29.4% reduction of SLNBs performed in patients with a Class 1A result relative to the baseline rate of 78.0% ( p  < .01). In patients ≥55 or ≥65-year-old, SLNB reduction was 32.3% ( p  < .01), 28.3% ( p  < .01), respectively. Overall, 85.3% of decisions relating to SLNB were influenced by 31-GEP results., Conclusion: In this prospective, multicenter study, clinicians demonstrated clinically meaningful use of the 31-GEP test to forego or pursue SLNB in patients with T1-T2 tumors resulting in a significant, risk appropriate decrease in SLNBs.

National guidelines recommend sentinel lymph node biopsy (SLNB) be offered to patients with > 10% likelihood of sentinel lymph node (SLN) positivity. On the other hand, guidelines do not recommend SLNB for patients with T1a tumors without high-risk features who have < 5% likelihood of a positive SLN. However, the decision to perform SLNB is less certain for patients with higher-risk T1 melanomas in which a positive node is expected 5%-10% of the time. We hypothesized that integrating clinicopathologic features with the 31-gene expression profile (31-GEP) score using advanced artificial intelligence techniques would provide more precise SLN risk prediction., Methods: An integrated 31-GEP (i31-GEP) neural network algorithm incorporating clinicopathologic features with the continuous 31-GEP score was developed using a previously reported patient cohort (n = 1,398) and validated using an independent cohort (n = 1,674)., Results: Compared with other covariates in the i31-GEP, the continuous 31-GEP score had the largest likelihood ratio (G 2 = 91.3, P < .001) for predicting SLN positivity. The i31-GEP demonstrated high concordance between predicted and observed SLN positivity rates (linear regression slope = 0.999). The i31-GEP increased the percentage of patients with T1-T4 tumors predicted to have < 5% SLN-positive likelihood from 8.5% to 27.7% with a negative predictive value of 98%. Importantly, for patients with T1 tumors originally classified with a likelihood of SLN positivity of 5%-10%, the i31-GEP reclassified 63% of cases as having < 5% or > 10% likelihood of positive SLN, for a more precise, personalized, and clinically actionable SLN-positive likelihood estimate., Conclusion: These data suggest the i31-GEP could reduce the number of SLNBs performed by identifying patients with likelihood under the 5% threshold for performance of SLNB and improve the yield of positive SLNBs by identifying patients more likely to have a positive SLNB., Competing Interests: John T. Vetto Employment: Gilead Sciences (I) Stock and Other Ownership Interests: Gilead Sciences (I), Roche/Genentech (I) Speakers' Bureau: Castle Biosciences Travel, Accommodations, Expenses: Castle Biosciences No other potential conflicts of interest were reported. John T. Vetto Employment: Gilead Sciences (I) Stock and Other Ownership Interests: Gilead Sciences (I), Roche/Genentech (I) Speakers' Bureau: Castle Biosciences Travel, Accommodations, Expenses: Castle Biosciences No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Gene expression profile (GEP) testing is now commercially available for metastatic risk prediction in patients with cutaneous squamous cell carcinoma (CSCC) and one or more high-risk factors. The purpose of this article is to provide an early framework for healthcare providers looking to integrate patient-specific tumor biology into their clinical practice using GEP testing. To develop a framework for clinical use, an expert panel was convened to identify CSCC management decision points where GEP testing may be immediately incorporated into practice until the definitive results of prospective trials become available. Based on their discussion, the expert panel focused on the areas of nodal evaluation, adjuvant radiation therapy, and follow-up and surveillance. The panel emphasized that GEP prognostic test results should not currently be used as a surrogate for standard of care treatment but as an additional data point when determining individualized management for patients with high-risk CSCC. Whenever possible, decisions on management plans for these patients should be developed with multidisciplinary input. J Drugs Dermatol. 2021;20:6(Suppl):s5-11. doi:10.36849/JDD.6068.

Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR)., Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC-MS., Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

Mass spectrometry-based quantitative proteomic analysis has proven valuable for clinical and biotechnology-related research and development. Improvements in sensitivity, resolution, and robustness of mass analyzers have also added value. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications where thousands of samples per month must be analyzed. To alleviate these issues, we developed a "cells-to-peptides" automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes 2 h to process 96 samples from cell pellets to the initiation of the tryptic digestion step and can process 384 samples in parallel. We measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography-mass spectrometry systems. The automated workflow was tested by preparing 96 Escherichia coli samples and quantifying over 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly multiplexed LC-MRM-MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications.

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C., (open access.)

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Objective: The aim of this study was to determine the impact of the results of the 12-gene DCIS Score assay on (i) radiotherapy recommendations for patients with pure ductal carcinoma in situ (DCIS) following breast-conserving surgery (BCS), and (ii) patient decisional conflict and state anxiety., Methods: Thirteen sites across the US enrolled patients (March 2014-August 2015) with pure DCIS undergoing BCS. Prospectively collected data included clinicopathologic factors, physician estimates of local recurrence risk, DCIS Score results, and pre-/post-assay radiotherapy recommendations for each patient made by a surgeon and a radiation oncologist. Patients completed pre-/post-assay decisional conflict scale and state-trait anxiety inventory instruments., Results: The analysis cohort included 127 patients: median age 60 years, 80 % postmenopausal, median size 8 mm (39 % ≤5 mm), 70 % grade 1/2, 88 % estrogen receptor-positive, 75 % progesterone receptor-positive, 54 % with comedo necrosis, and 18 % multifocal. Sixty-six percent of patients had low DCIS Score results, 20 % had intermediate DCIS Score results, and 14 % had high DCIS Score results; the median result was 21 (range 0-84). Pre-assay, surgeons and radiation oncologists recommended radiotherapy for 70.9 and 72.4 % of patients, respectively. Post-assay, 26.4 % of overall recommendations changed, including 30.7 and 22.0 % of recommendations by surgeons and radiation oncologists, respectively. Among patients with confirmed completed questionnaires (n = 32), decision conflict (p = 0.004) and state anxiety (p = 0.042) decreased significantly from pre- to post-assay., Conclusions: Individualized risk estimates from the DCIS Score assay provide valuable information to physicians and patients. Post-assay, in response to DCIS Score results, surgeons changed treatment recommendations more often than radiation oncologists. Further investigation is needed to better understand how such treatment changes may affect clinical outcomes.

In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7-21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28-38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.

Background and Objectives: Twenty percent of breast cancers are ductal carcinoma in situ (DCIS), with 15-60% having a local recurrence (LR) after surgery. Radiotherapy reduces LR by 50% but has not impacted survival. The validated Oncotype DX(®) 12-gene assay (DCIS Score) provides individualized 10-year LR estimates. This is the first study to assess whether DCIS Score impacts physicians' recommendations for radiation., Methods: Ten sites enrolled women (9/2012-2/2014) with DCIS eligible for breast-conserving therapy, excluding patients with invasive carcinoma and planned mastectomy. Prospective data collected included clinicopathologic factors, DCIS Score assay, and treatment recommendation before and after the assay result was known., Results: In 115 patients (median age: 61 years; 74.8% postmenopausal), median DCIS size was 8 mm; 20% were nuclear grade 1, 46.1% grade 2; 64.4% reported necrosis. 86.1% were ER+, 79.1% PR+ (immunohistochemistry assay). Median DCIS Score: 29 (range: 0-85). Pre-assay, 73% (95%CI: 64.0-80.9%) had radiotherapy recommendations vs. 59.1% (95%CI: 49.6-68.2%) post-assay (P= 0.008). Physicians rated DCIS Score as the most impactful factor in planning treatment., Conclusions: The radiotherapy recommendation changed from pre-assay to post-assay 31.3% (95%CI: 23.0-40.6%) of the time--a clinically significant change. This study supports the clinical utility of the DCIS Score and indicates that the test provides additional, individualized information on LR risk., (© 2015 Wiley Periodicals, Inc.)

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different β-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

The principal cause of a high mortality rate in mesenteric vein thrombosis (MVT) is a delay in diagnosis. Recent data indicate that the mortality rate is decreasing owing to earlier diagnosis and anticoagulation. The authors examined the treatment profile of MVT to see how the increased use of imaging and early anticoagulation has impacted this process. They retrospectively analyzed the treatment paradigm with acute MVT at one institution over a 10-year period. Twenty-three patients were identified. Data were analyzed using chi-squares and Student's t tests. Twenty-three patients (11 men and 12 women with an average age of 51.74 +/-14.8 years) were identified with acute MVT between the years of 1993 and 2003. Five patients had splenic vein thrombosis, 17 had superior mesenteric vein thrombosis, 1 had inferior mesenteric vein thrombosis, and 12 had portal vein thrombosis. Nine patients had combination mesenteric vein segment thrombosis. Thrombolytics were utilized in a total of 6 patients. Four of the 6 patients in whom lytics were utilized had combined mesenteric vein thrombosis; however, these 4 patients did not require surgical intervention. There was no significant difference in length of hospital stay between patients taking lytics versus patients treated with traditional anticoagulation with heparin (p = 0.291). A hypercoagulable state was identified in 66.7% of the patients. Four patients required surgical intervention. The overall mortality rate was 8.7% (2 of 23). The use of thrombolytics was associated with a significant mortality (p = 0.04). The use of antibiotics made no difference in mortality (p = 0.235), nor did antibiotic use influence length of hospitalization (p = 0.192). MVT is relatively rare, and often the delay in diagnosis increases the mortality rate. In the majority of cases prompt anticoagulation will preserve bowel viability and decrease mortality and morbidity rates. The majority of patients do not need surgery. There is a marked increase in mortality rate when these patients progress to surgical intervention. An increased awareness and early diagnosis has led to decreased morbidity and mortality rates.

Currently, 1 per cent isosulfan blue dye and technetium-99-labeled sulfur colloid (SC) are used in lymphatic mapping (LM). Several reports have suggested that the incidence of adverse drug reactions (ADRs) during LM is high. We report our experience with LM for solid neoplasms in order to determine the incidence and risk factors for development of ADRs. Seven hundred fifty-three patients (90% women, mean age 57) underwent LM with blue dye alone or in combination with SC from 1998 to 2004. The most common malignancy was breast cancer (83%). One hundred ten patients (14%) had injection of both mapping agents. Most patients (87%) underwent intraparenchymal injection of LM agent. Eight patients (1.1%) had an ADR during LM; none had prior exposure to LM. Of these, 7 had limited reactions (mostly blue hives) that quickly resolved. One patient (0.1%) developed anaphylaxis. The ADR incidence in patients with a sulfa allergy was not significantly different than that in patients without a sulfa allergy (3.4 vs 1%, P = 0.12). No risk factors for development of ADR were identified. Overall, the incidence of ADR during LM is low. Patients with sulfa allergies and prior exposure to LM did not demonstrate an increased incidence of ADR. Anaphylaxis, though rare, can occur during LM.

Sendai virus (SeV) infection causes the transcriptional induction of many cellular genes that are also induced by interferon (IFN) or double-stranded RNA (dsRNA). We took advantage of various mutant cell lines to investigate the putative roles of the components of the IFN and dsRNA signaling pathways in the induction of those genes by SeV. Profiling the patterns of gene expression in SeV-infected cells demonstrated that Toll-like receptor 3, although essential for gene induction by dsRNA, was dispensable for gene induction by SeV. In contrast, Jak1, which mediates IFN signaling, was required for the induction of a small subset of genes by SeV. NF-kappaB and interferon regulatory factor 3 (IRF-3), the two major transcription factors activated by virus infection, were essential for the induction of two sets of genes by SeV. As expected, some of the IRF-3-dependent genes, such as ISG56, were more strongly induced by SeV in IRF-3-overexpressing cells. Surprisingly, in those cells, a number of NF-kappaB-dependent genes, such as the A20 gene, were induced poorly. Using a series of cell lines expressing increasing levels of IRF-3, we demonstrated that the degree of induction of A20 mRNA, upon SeV infection, was inversely proportional to the cellular level of IRF-3, whereas that of ISG56 mRNA was directly proportional. Thus, IRF-3 can suppress the expression of NF-kappaB-dependent genes in SeV-infected cells.

Lymphatic mapping (LM) and sentinel lymph node biopsy (SLNB) have become widely accepted in the setting of breast conservation surgery. We hypothesized that LM can be extended to women undergoing total mastectomy, being technically feasible, yielding highly accurate and sensitive results, improving axillary staging, and reducing postoperative morbidity. Between 1995 and 2003, 99 women (mean age 59 years, range 34-87) underwent 100 mastectomies with LM using blue dye alone. Fifty-nine operations (60%) were followed by a completion axillary lymph node dissection (ALND). Ninety per cent of patients had invasive carcinoma; 10 per cent had in situ carcinoma. Mean tumor size was 2.5 cm (range 0.3-8 cm). One hundred fifty-nine sentinel nodes (SNs) (mean 1.65, range 1-5) were successfully identified in 96 (96%) axillae. Twenty-five (25%) sentinel nodes revealed nodal metastases. Five of 25 (20%) SNs had micrometasteses. Three patients had a false-negative SN, yielding a sensitivity of 91 per cent. The accuracy of LM was 97 per cent. No patient who underwent SLNB alone developed lymphedema, axillary seroma formation, infection, or restricted arm movement. This was contrasted with patients undergoing ALND, where 10 (16%) developed lymphedema and 2 (3%) developed an infection. Ten (25%) patients developed axillary paresthesias after SNB compared with 47 (78%) patients after ALND (P < 0.0001). LM in the setting of mastectomy is accurate and sensitive. This technique improves axillary staging and decreases morbidity. Patients who are not candidates for breast conservation should be offered LM and SLNB at the time of mastectomy.

Serosanguinous drainage after mastectomy and axillary lymph node dissection has traditionally been treated with the temporary use of closed suction drainage catheters. Use of drainage catheters is associated with wound infection, discomfort, nerve injury, and impaired arm movement. Commercially produced fibrin sealant has been proposed to reduce postoperative serosanguinous collections. We hypothesized that the intraoperative application of low-dose (2-5 cm3) fibrin sealant would reduce serosanguinous drainage and allow earlier removal of closed suction drainage catheters after operation for breast cancer. Fifty-five women with known breast cancer underwent either total mastectomy, modified radical mastectomy, or isolated level I and II axillary lymph node dissection. Twenty-six patients were treated with fibrin sealant and 29 served as control subjects. The application of fibrin sealant resulted in a significant reduction in overall duration catheters were needed (7 vs 8.3 days; P = 0.05). More importantly fibrin sealant reduced the time until 24-hour drain output was less than 30 cm3 (4.9 vs 6.2 days). Additionally fibrin sealant application resulted in a 60 per cent reduction in overall drainage amount after total mastectomy and a 32 per cent reduction after modified radical mastectomy. The application of fibrin sealant after axillary lymph node dissection did not decrease overall drainage amount. In conclusion fibrin sealant reduces serosanguinous drainage after total mastectomy and modified radical mastectomy and may allow earlier removal of closed suction drainage catheters.

Hypothesis: Sentinel node (SN) biopsy for breast cancer enhances staging sensitivity, often demonstrating only micrometastases (<2 mm) or isolated, keratin-positive cells. When SN metastasis is present, the value of additional axillary dissection is unclear and not all patients benefit from axillary lymph node dissection (ALND)., Design: Prospective cohort study, median 32-month follow-up., Setting: Multidisciplinary breast cancer centers., Patients: Forty-six women having SN metastases diagnosed between May 1, 1996, and September 1, 2001, who refused ALND or were recommended to omit ALND owing to serious comorbid conditions., Interventions: Isosulfan blue dye-directed SN biopsy. Axillary lymph node dissection was not performed. Standard breast irradiation was given. Adjuvant systemic therapy was provided as determined by an oncologist. Interval clinical evaluation was performed., Main Outcome Measure: Axillary and systemic failure rates., Results: Mean patient age was 61.6 years (age range, 36-92 years). Mean tumor size was 1.65 cm (range, 0.4-5.5 cm). Thirty-five (76%) of 46 tumors were ductal carcinomas and 39 (87%) of 45 were estrogen receptor-positive. A mean of 2.6 SNs were identified (median, 2; range, 1-7). Thirty-nine patients (85%) had a single positive SN; the remaining 7 patients (15%) had 2 positive SNs. Seven patients (15%) had macrometastases (>2 mm); 16 (35%) had micrometastases (<2 mm); and 23 (50%) had cellular metastases. Only 16 positive SNs (35%) were seen on hematoxylin-eosin staining, while 30 SNs (65%) had positive immunohistochemical staining. There have been no axillary recurrences. One patient (2%) developed distant metastases during follow-up (range, 4-61 months)., Conclusions: Patients with SN metastases who did not have ALND had a low incidence of regional failure. To confirm this observation, we suggest that patients with SN metastases are ideal candidates for trials evaluating the necessity of ALND.

Purpose: Sentinel lymph node (SLN) biopsy is a widely accepted method for staging breast cancer and melanoma, and it has recently been proposed as a means of improving staging in colorectal cancer. However, lymphatic mapping in colorectal cancer has been plagued by studies demonstrating high false-negative rates. The purpose of this study was to evaluate possible mechanisms for high false-negative rates after SLN biopsy in colorectal cancer. We hypothesized that poor accuracy may be due to bulky tumor or complete replacement of lymph nodes by tumor., Patients and Methods: Patients with colorectal adenocarcinoma underwent standard colorectal resection with lymphatic mapping. At operation, 1 mL of isosulfan blue dye was injected at the tumor site, using either an in vivo or an ex vivo technique. Routine pathological evaluation was performed. The sentinel node was examined by hematoxylin and eosin stains, and if these results were negative, by cytokeratin immunohistochemistry. The patient's age, operation type, tumor stage, tumor diameter, method of SLN detection, presence of palpable nodes, and pathological description of nodes completely replaced by tumor were recorded., Results: Fifty patients (mean age, 62.8, 50% men) undergoing colorectal cancer resection underwent 51 lymphatic mapping procedures. Right- and left-sided colorectal resections were almost equally distributed (48% vs 42%). SLNs were successfully identified in 47 of 51 specimens (92%). The mean number of SLNs obtained from each specimen was 1.5 (range, 1-5). Routine pathological evaluation demonstrated lymph node metastasis in 20 of the 47 patients (43%) who had an SLN identified. The SLN was positive for metastasis in 10 of these 20 patients (50%). Ten of 20 patients with metastatic disease had a negative SLN, resulting in a false-negative rate of 50%. The false-negative rate was significantly higher in patients undergoing left-sided procedures versus right-sided procedures. Differences among gender, tumor stage, tumor diameter, method of SLN detection, presence of palpable nodes, and pathological description of nodes completely replaced by tumor were not associated with a higher false-negative rate., Discussion: Identification of the SLN in colorectal cancer is technically possible in more than 90% of patients. However, SLN status correlates poorly with the true nodal status of the colorectal cancer, and the false-negative rate is 50%. This high false-negative rate is not clearly explained by extensive tumor burden, and it was also independent of gender, tumor stage, and type of lymphatic mapping technique. However, staging accuracy was lower in patients who underwent left-sided colorectal resection. Further studies are needed to clarify the limitations of lymphatic mapping in colorectal cancer.

Background: Dye-directed sentinel node biopsy (SNB) for breast cancer provides accurate staging with low morbidity, but for tumors distant from the axilla, its use has been questioned., Hypothesis: Can preoperative breast lymphoscintigraphy (BL) applied selectively to medial hemisphere tumors predict a subset of patients who may not require surgical staging of the axilla?, Design: Prospective cohort study., Setting: Tertiary, multidisciplinary breast center., Patients: Thirty-two women with breast tumors located in the medial hemisphere (30) or inframammary crease (2)., Intervention: Peritumoral injection of 500 microCi of technetium Tc 99m sulfur colloid and biplanar imaging. Nonpalpable lesions were localized with ultrasound or mammography. At the time of definitive breast surgery, isosulfan blue dye-directed SNB was performed. Axillary dissection was performed when the SN contained a tumor or could not be identified., Main Outcome Measures: Regional nodal basins identified by BL; success rate of SNB., Results: Preoperative BL demonstrated axillary drainage in 28 patients (88%); 2 patients (6%) had isolated internal mammary radionuclide uptake, and 2 patients had no nodal uptake. Dye-directed axillary SNB succeeded in 27 (87%) of 31 patients, including both patients with failed BL. Breast lymphoscintigraphy had predicted isolated internal mammary drainage in 2 of 4 patients whose SNs could not be identified. Metastases were found in 5 patients (16%)., Conclusions: Axillary radionuclide uptake predicts but does not augment dye-directed SN identification. In those few patients with isolated internal mammary drainage, BL may obviate the need for surgical staging of the axilla.

Background: Although more than 90% of the morbidity and mortality from localized cutaneous melanoma occurs in the first decade after initial surgical treatment, melanoma can recur after a 10-year disease-free interval (DFI) with fatal consequences. We reviewed our melanoma data base of more than 8,500 prospectively acquired patients to identify clinicopathological factors that affect the type, rate of occurrence, and outcome of disease recurring 10 years or more after surgical treatment of primary cutaneous melanoma., Methods: From 1971 to 1997, 1907 melanoma patients treated at our cancer center reached or presented with a DFI of 10 years or more after surgical treatment of clinically localized melanoma. Of these, 217 (11%) patients had recurrences (mean DFI, 182 months). The sites of recurrence were local/in-transit in 26 (12%) patients, regional lymph nodes in 101 (47%) patients, and distant sites in 90 (41%) patients., Results: Univariate and multivariate analysis, using patient age and sex, type of initial treatment, and the site, Breslow thickness, and Clark level of the initial tumor, showed that the type of treatment for the primary tumor was a significant (P = .0005) prognostic factor in the development of late nodal recurrence. Of the 217 patients who had recurrences, 172 (79%) had undergone wide local excision for their primary melanoma, and 45 (21%) had undergone wide local excision plus elective lymph node dissection (ELND). The rates of nodal recurrence were 53% (92 of 172) and 20% (9 of 45), respectively, a significant (P = .0001) difference. When all patients with a DFI of 10 years or more were stratified by type of initial treatment, the ELND group demonstrated a significant improvement in disease-free survival and overall survival., Conclusions: The risk of late-recurring nodal disease increases and the chance of long-term survival decreases when wide local excision is performed without ELND. With the advent of sentinel lymphadenectomy, ELND can be selectively performed only for those nodal basins with occult tumor cells, thereby decreasing operative morbidity but allowing identification and early removal of nodal micrometastases.

Intraoperative lymphatic mapping and sentinel lymphadenectomy (LM/SL) has been demonstrated to provide sensitive axillary staging for breast cancer. LM/SL has a steep learning curve, and factors associated with unsuccessful LM/SL are not well known. Two hundred sixty patients with breast carcinoma and clinically negative axillae underwent injection of about 5 cm3 of isosulfan blue dye (Lymphazurin, US Surgical Corp, Norwalk, CT) into breast tissue surrounding a cancer or biopsy site. After 5 minutes of breast compression, blue-stained lymph nodes were sought. In 47 patients, no blue nodes were detected; a standard axillary dissection was performed. All 47 patients were women with a mean age of 56 years (range, 34-80). Ductal carcinoma was most common (91.5%). Mean tumor size was 1.99 cm. Axillary dissection yielded a mean of 15.8 lymph nodes (range, 6-35). Sixteen patients (34%) had positive lymph nodes (mean, 7.6; median, 6; range, 1-24). Factors associated with LM/SL difficulty include surgeon inexperience, medial hemisphere primary location, extensive axillary metastases, and extranodal invasion. Inability to identify a sentinel node in a clinically negative axilla is a risk factor for extensive axillary tumor burden. Axillary dissection should be performed for patients with unsuccessful LM/SL, particularly those with lateral hemisphere primaries.

Purpose: To evaluate the feasibility, accuracy, and reproducibility of intraoperative lymphatic mapping and sentinel lymphadenectomy (IOLM/SL) in the staging of breast cancer patients in a community managed care setting., Patients and Methods: One hundred forty-five patients with primary breast cancer were prospectively studied over a 26-month period. They underwent vital dye injection at their primary breast cancer site. Lymphatic channels were traced to the sentinel lymph node, which was excised, serially sectioned, and examined. A level I and II axillary lymph node dissection and definitive breast surgery were then performed., Results: Sentinel nodes were identified in 103 of 145 procedures (71.0%). Sentinel and nonsentinel lymph nodes were concordant in 100 of 103 cases (97.1%). Three patients (9.7%) had falsely negative sentinel nodes; there were none in the last 80 patients. Of 28 positive sentinel nodes, 12 (42.9%) represented the only tumor-containing node within the axilla. Sentinel nodes were significantly more likely to contain tumor than nonsentinel nodes (33/50, 66.0% vs 54/467, 11.6%, P < 0.0001). IOLM/SL identified more micrometastases (< 2 mm) than standard axillary lymph node dissection (13/33, 39.6% vs 4/177, 2.2%, P < 0.001). Nine of 42 patients (21.4%) whose sentinel node could not be identified had five or more nodal metastases. Two of six patients with presumed Tis primaries had nodal metastases., Discussion: IOLM/SL accurately identifies the sentinel lymph node(s) most likely to contain metastatic disease. A procedural learning curve was present. An unsuccessful IOLM/SL was a risk factor for considerable nodal metastases. IOLM/SL with a tumor-free sentinel node may obviate a formal axillary lymph node dissection. The technique was feasible, economical, and reproducible within the context of a community managed care facility, while not placing exacting demands on operating room, pathology, or nuclear medicine personnel.

We attempted to show that surgical treatment of breast cancer, including axillary lymph node dissection with or without concomitant partial mastectomy (ALND), simple mastectomy (SM), and modified radical mastectomy (MRM) can be performed safely in an outpatient setting. The records of 100 consecutive women undergoing definitive breast cancer surgery by the authors between August 1994 and July 1996 were retrospectively reviewed. Average age was 54 +/- 10 years. Fifty patients were discharged the day of surgery, 44 were hospitalized, and 6 remained 2 or more days postoperatively. Outpatients were more likely to have undergone ALND or SM (42 versus 23 procedures) and more often completed surgery in the morning (36 versus 12); P < 0.05. Eight patients of 35 with MRM were discharged the same day. One patient was readmitted with a wound infection. There were no major complications or deaths. Ninety-four per cent of patients were discharged within 23 hours of surgery; half were discharged the same day. No complications occurred in outpatients, and there were no readmissions. For patients admitted overnight, no complications were detected during the overnight hospital stay. In conclusion, breast cancer surgery, from ALND to SM or MRM, can be safely and comfortably performed on an outpatient basis.

Objective: To determine if breast-conserving therapy (BCT) consisting of segmentectomy, axillary lymph node dissection, and postoperative irradiation is a feasible approach to breast cancer in younger women, whose breast tissue is dense and whose tumors can be difficult to detect and successfully excise., Design and Patients: We studied BCT in 59 women 35 years old or younger (mean age, 31.7 years) treated for breast cancer since 1982. Ninety percent of their cancers were palpable; 44% were not visible by mammography. Most (93%) had T1 or T2 lesions (< or = 5 cm). Invasive ductal carcinoma was the predominant histologic diagnosis (68%)., Results: Segmentectomy with axillary dissection was the initial operative procedure for 39 (66%) of the 59 patients; of these, 21 (54%) had microscopically positive segmentectomy margins. Nine patients (23%) with diffusely positive segmentectomy margins and four patients (13%) with local recurrences after BCT required conversion to mastectomy. Three patients (8%) underwent reexcision to achieve negative margins. The 39 patients required a total of 22 additional surgical procedures for local control. Thirty-three (56%) of the 59 patients underwent mastectomy during the mean 68-month follow-up period; 20 (34%) underwent mastectomy as the initial definitive treatment. Reasons for primary mastectomy included multifocality or multicentricity (35%), large tumor size (30%), patient preference (15%), and occult primary tumor (10%). During the same time period, 474 (64%) of 745 women older than 35 years underwent BCT as treatment of breast cancer. Two percent required conversion to mastectomy and 1% required repeated excision. Twenty-four patients (5%) required mastectomy for local recurrence after BCT. After excluding mastectomies performed because of patient preference, significantly fewer older women required mastectomy to achieve local control (21% vs 50%, P < .001)., Conclusions: Breast-conserving therapy is significantly more difficult in younger women despite surgeon and patient commitment. Patients and physicians should be encouraged to consider BCT but should be aware of the potential difficulty in obtaining adequate local control and the possible need for additional operative procedures.

Objective: The authors report the feasibility and accuracy of intraoperative lymphatic mapping with sentinel lymphadenectomy in patients with breast cancer., Summary Background Data: Axillary lymph node dissection (ALND) for breast cancer generally is accepted for its staging and prognostic value, but the extent of dissection remains controversial. Blind lymph node sampling or level I dissection may miss some nodal metastases, but ALND may result in lymphedema. In melanoma, intraoperative lymph node mapping with sentinel lymphadenectomy is an effective and minimally invasive alternative to ALND for identifying nodes containing metastases., Methods: One hundred seventy-four mapping procedures were performed using a vital dye injected at the primary breast cancer site. Axillary lymphatics were identified and followed to the first ("sentinel") node, which was selectively excised before ALND., Results: Sentinel nodes were identified in 114 of 174 (65.5%) procedures and accurately predicted axillary nodal status in 109 of 114 (95.6%) cases. There was a definite learning curve, and all false-negative sentinel nodes occurred in the first part of the study; sentinel nodes identified in the last 87 procedures were 100% predictive. In 16 of 42 (38.0%) clinically negative/pathologically positive axillae, the sentinel node was the only tumor-involved lymph node identified. The anatomic location of the sentinel node was examined in the 54 most recent procedures; ten cases had only level II nodal metastases that could have been missed by sampling or low (level I) axillary dissection., Conclusions: This experience indicates that intraoperative lymphatic mapping can accurately identify the sentinel node--i.e., the axillary lymph node most likely to contain breast cancer metastases--in some patients. The technique could enhance staging accuracy and, with further refinements and experience, might alter the role of ALND.

Background: Although breast-conserving therapy (tumor excision, axillary node dissection, and postoperative radiation) for women with breast cancer yields survival and local recurrence rates comparable with those of modified radical mastectomy, studies suggest that postoperative radiation leads to capsular contractures and poor cosmesis in patients with breast implants., Methods: The authors followed 20 women in whom breast cancer developed after augmentation mammoplasty (14 subcutaneous implants and 6 retromuscular implants). Average age at diagnosis was 52 years (range, 34-72 years). Most (55%) of the patients had tumors in the upper outer quadrant. Fifteen lesions were palpable and five were nonpalpable. All tumors were excised using wide margins that attempted to include a rim of normal breast tissue. Three patients had microscopically positive margins. The predominant histology was ductal adenocarcinoma (85%). The mean greatest tumor dimension was 1.43 cm; 75% were T1 lesions. Levels I and II axillary lymph node dissection revealed metastases in five patients. After surgery, six patients received systemic chemotherapy, and all patients received 4500-5000 cGy of tangential photon radiation delivered to the whole breast, plus a 1400-2100 cGy boost delivered to the tumor site using photon radiation, electron radiation, or iridium 192 implantation., Results: At a median follow-up of 3.8 years (range, 6 months to 9.3 years), there were no local recurrences; however, in two patients distant metastases developed. Seventeen (85%) of the twenty patients had good or excellent cosmetic results as determined by the degree of capsular contracture, breast shape and appearance, and the presence of skin changes., Conclusions: The authors conclude that breast-conserving therapy is a cosmetically acceptable therapeutic option for women in whom breast cancer develops after augmentation mammoplasty.

Objective and Design: Some surgeons consider excisional biopsy with gross negative margins to be adequate surgical therapy for breast carcinomas, if followed by axillary dissection and radiation. To test our hypothesis that breast carcinoma necessitates planned operation, we reviewed the incidence of residual cancer tissue (RCT) and the significance of positive margins following excisional breast biopsy and segmentectomy. SETTING, PATIENTS, AND INTERVENTION/OUTCOME MEASURES: Using the clinical database of our multidisciplinary cancer center, we examined the tumor status of segmentectomy specimens from 375 patients treated for breast carcinoma during the past 10 years. All patients underwent excisional biopsy of the tumor mass before definitive treatment with segmentectomy and axillary dissection. Median follow-up was 32 months., Results: The 284 patients (76%) whose segmentectomy specimens contained residual tumor (RCT-positive patients) had a larger median tumor diameter than RCT-negative patients (2 vs 1 cm, P < .01). Patients with tumor-positive axillary lymph nodes were more likely to be RCT positive (P < .001). Tumors of RCT-positive patients were more frequently identified by physical examination, whereas those of RCT-negative patients were more frequently identified by mammography (P < .001). Overall recurrence rate was 7% (26/384). Recurrence-free survival rates were statistically related to tumor status of the segmentectomy margins (P < .025) but not to RCT in the segmentectomy specimen., Conclusion: Diagnostic breast biopsy is not a substitute for planned excision to remove all malignant tissue. Anything less than a preconceived surgical procedure may leave a significant amount of malignant tissue.