Degnim, Amy C.

Background: Prepectoral implant-based breast reconstruction has gained popularity because of advantages over the subpectoral technique. Acellular dermal matrix use with implant-based breast reconstruction has become common because of its perceived superior aesthetic outcome. Matrices are expensive, however, and recent evidence has pointed to several potential complications. This article reports a series of prepectoral implant-based breast reconstructions with and without acellular dermal matrix and compared their outcomes. Methods: This is a single-surgeon retrospective review of patients who underwent staged prepectoral implant-based breast reconstruction following nipple-sparing mastectomy over two periods. Patients with well-perfused mastectomy skin flaps with a homogeneous thickness underwent reconstruction with acellular dermal matrix initially. On evolution of the practice, it was not used. Patient demographics, operative data, and complications were analyzed. Aesthetic outcome was measured by the BREAST-Q survey and the Aesthetic Item Scale. A cost analysis was also performed. Results: Forty patients were included (acellular dermal matrix group, n = 19; non–acellular dermal matrix group, n = 21). The nonmatrix group had one case (5 percent) of seroma and one case (5 percent) with hematoma; there were none in the acellular dermal matrix group. Average BREAST-Q and Aesthetic Item Scale scores were 82.3 versus 81.6 (p = 0.954) and 20.98 versus 20.43 (p = 0.640) for the matrix and nonmatrix groups, respectively. The direct cost savings for the authors’ institution over 1 year if matrix was not used in all cases of implant-based breast reconstruction would be estimated at $3,105,960 to $6,211,920 for unilateral and bilateral cases, respectively, for Medicare reimbursement. Conclusions: With adequate patient selection, acellular dermal matrix is not always required during two-stage prepectoral implant-based breast reconstruction for good aesthetic outcomes. The economic burden on patients and the health care system could be lessened with selective matrix use. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

AbstractPurpose:Patients with HER2+ breast cancer benefit from trastuzumab-containing regimens with improved survival. Adaptive immunity, including cytotoxic T-cell and antibody immunity, is critical to clinical efficacy of trastuzumab. Because Th cells are central to the activation of these antitumor effectors, we reason that HER2 patients treated with trastuzumab may benefit by administering vaccines that are designed to stimulate Th-cell immunity.Patients and Methods:We developed a degenerate HER2 epitope–based vaccine consisting of four HLA class II–restricted epitopes mixed with GM-CSF that should immunize most (≥84%) patients. The vaccine was tested in a phase I trial. Eligible women had resectable HER2+ breast cancer and had completed standard treatment prior to enrollment and were disease free. Patients were vaccinated monthly for six doses and monitored for safety and immunogenicity.Results:Twenty-two subjects were enrolled and 20 completed all six vaccines. The vaccine was well tolerated. All patients were alive at analysis with a median follow-up of 2.3 years and only two experienced disease recurrence. The percent of patients that responded with augmented T-cell immunity was high for each peptide ranging from 68% to 88%, which led to 90% of the patients generating T cells that recognized naturally processed HER2 antigen. The vaccine also augmented HER2-specific antibody. Immunity was sustained in patients with little sign of diminishing at 2 years following the vaccination.Conclusions:Degenerate HLA-DR–based HER2 vaccines induce sustainable HER2-specific T cells and antibodies. Future studies, could evaluate whether vaccination during adjuvant treatment with trastuzumab-containing regimens improves patient outcomes.

Background Single-stage direct-to-implant (DTI) breast reconstruction can offer several potential benefits. Subpectoral DTI reconstruction can present with animation deformity and pectoralis muscle spasm. To potentially avoid these complications, surgeons have attempted prepectoral placement for DTI; however, the benefits of this approach are mostly unknown. We evaluated the outcomes of DTI between prepectoral and subpectoral placement. Methods This was a retrospective review of patients who underwent immediate DTI breast reconstruction (prepectoral vs subpectoral) between 2011 and 2018. Demographics, clinical characteristics, complications, and patient-reported outcomes (BREAST-Q) were compared. Results Thirty-three patients (55 breasts) underwent prepectoral DTI, and 42 patients (69 breasts) underwent subpectoral DTI. Demographics were similar among groups. The number of breasts with preoperative ptosis lower than grade 2 was not significantly different between groups (29.1% vs 26.1%; P = 0.699). Median follow-up was 20.3 and 21 months in the prepectoral and subpectoral groups, respectively. Average mastectomy weight was 300 g (180–425 g) and 355 g (203–500 g). Average implant size was 410 cc (330–465 cc) and 425 cc (315–534 cc) in the prepectoral and subpectoral groups, respectively. Alloderm was used in all reconstructions. Total numbers of complications were 4 (7.2%) and 8 (11.6%) in the prepectoral and subpectoral groups, respectively (P = 0.227). BREAST-Q demonstrated mean patient satisfaction was high and similar among groups (75 and 73.9, P = 0.211). Conclusions Based on these results, we believe prepectoral DTI is safe, reliable, and a promising reconstructive option for selected patients, with equivalent results to other reconstructive options. Our present treatment recommendations are for patients who wish to maintain the same breast size and have minimal or no breast ptosis.

Abstract Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92 % of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Background: Surgical-site infection after implant-based breast reconstruction adversely affects surgical outcomes and increases health care costs. This 11-year case-control study examines risk factors specific for surgical-site infection after immediate tissue expander/implant-based breast reconstruction. Methods: The authors performed a retrospective review to identify all consecutive patients with breast implant infections between 2006 and 2016. Patients who developed surgical-site infection after immediate tissue expander/implant-based breast reconstruction were included. Surgical-site infection was defined using the Centers for Disease Control and Prevention criteria; specifically, infections requiring hospital admission, intravenous antibiotics, or surgical intervention were included. The authors matched a control patient to each infection case by patient age and date of surgery. Patient demographics, medical comorbidities, and perioperative surgical variables were examined. Univariate and multivariable conditional logistic regression models were constructed. Results: A total of 270 breasts in 252 patients were evaluated. On multivariate analysis, patients with a higher body mass index (OR, 1.1 per 1 body mass index point increase; 95 percent CI, 1.0 to 1.2; p = 0.02), hypertension (OR, 6.5; 95 percent CI, 1.9 to 22.3; p = 0.002), neoadjuvant chemotherapy (OR, 2.6; 95 percent CI, 1.0 to 6.3; p = 0.04), axillary lymph node dissection (OR, 7.1; 95 percent CI, 1.7 to 29.2; p = 0.006), seroma formation (OR, 15.34; 95 percent CI, 3.7 to 62.5; p = 0.0001), and wound healing complications (OR, 23.91; 95 percent CI, 6.1 to 93.4; p < 0.0001) were significantly associated with surgical-site infection. Conclusions: Women with obesity, women with hypertension, and those treated with neoadjuvant chemotherapy are at increased risk of surgical-site infection. Further risks are also associated with postoperative seroma and wound complications. This may help patient selection and counseling, adjusted based on risk factors regarding complications of immediate implant-based breast reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Background and objectives: Differences in patient anatomy and physiology exist between young and older patients undergoing breast reconstruction after mastectomy. Breast cancer has been described as being more aggressive, more likely to receive radiation, contralateral mastectomy, as well as bilateral reconstruction in young patients. Our purpose is to report long-term experience on two-staged implant-based breast reconstruction (IBR) in young females, with complication sub-analysis based on obesity and adjuvant radiation. Materials and Methods: Retrospective chart review of all consecutive young patients who underwent two-staged IBR at our institution, between 2000 and 2016, was performed. Patients between 15 and 40 years old with least 1-year follow-up were included. Univariate logistic regression models and receiver operating characteristic (ROC) curves were created. Results: Overall 594 breasts met our inclusion criteria. The mean age was 34 years, and the median follow-up was 29.6 months. Final IBR was achieved in 98% of breasts. Overall, 12% of breasts had complications, leading to explantations of 5% of the devices. Adjuvant radiation was followed by higher rates of total device explantations (p = 0.003), while obese patients had higher rates of total complications (p < 0.001). For each point increase in BMI, the odds of developing complications increased 8.1% (p < 0.001); the cutoff BMI to predict higher complications was 24.81 kg/m2. Conclusions: This population demonstrates high successful IBR completion and low explantation rates. These data suggest that obese women and those with planned adjuvant radiation deserve special counseling about their higher risk of complications.

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Abstract Existing risk models underperform among women who have undergone a benign breast disease (BBD) biopsy (&gt; one million performed annually in the US) and with respect to estimating the risk of aggressive BCs. We've shown that molecular pathologic analysis of BBD biopsies can improve individual risk prediction, compared with standard risk models relying on self-reported factors, and provide insights into mechanisms mediating BC risk. BC risk factors such as obesity and ethanol use are proposed to increase production of cytokines and chemokines, resulting in chronic inflammation, and leading to production of DNA damaging free radicals and growth factors (e.g., VEGF, IGFs) that activate pro-carcinogenic pathways (e.g. NF-KB and JAK/STAT). The effects of most BC risk factors on immunity are poorly understood. Further, whereas experimental models implicate immunity throughout carcinogenesis, knowledge of immune markers and mechanisms related to BC development among women is limited. Therefore, we aim to define tissue immune cell content throughout BC development, and particularly at the earliest stages, to improve risk assessment and discover immune-based prevention strategies. Our study combines novel resources and technologies to define the immune landscape in: 1) normal breast tissues in relation to BC risk factors, 2) BBD with respect to BC risk, and 3) invasive BC in relation to neoantigen expression and molecular subtype. We will perform NanoString GeoMxTM Digital Spatial Profiling (DSP) to quantitatively map expression of key immune proteins in healthy breast tissues donated to the Komen Tissue Bank (KTB) in relation to BC risk factors and in BBD biopsies from two cohorts to predict BC risk. We will re-analyze top prognostic markers in BBD biopsies using Vectra multiplex IF staining with machine learning algorithms to refine how immune cell content and organization impacts prognosis, and assess critical immune pathways related to BBD progression with the NanoString PanCancer IO 360 panel, which provides RNA expression of 770 immune genes (13 signatures). Finally, we will evaluate immune responses in BCs categorized for predicted neoantigen load and underlying mutation type with our novel bioinformatics pipeline that enables accurate prediction of MHC class I and class II missense, fusion and frameshift mutation neoantigens generated through faulty DNA repair, aberrant DNA and RNA splicing, insertions, and deletions. Our project demonstrates a novel approach to defining the immune processes in early stages of BC development which may improve BC risk prediction and prevention. We present a plan to comprehensively analyze immunity throughout BC development and define changes that accompany transitions from normal to BBD and BBD to BC. Citation Format: Doug Hinerfeld, Keith Knutson, Derek Radisky, E. Aubrey Thompson, Yan Asmann, Kim McCoy, Amy Degnim, Jodi Carter, Stacey Winham, Michele Cote, Jeroen van der Laak, Mark Sherman. Analysis of the immune microenvironment to advance breast cancer risk prediction and prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 651.

Abstract Background: Macrophages are well-established components of the inflammatory repertoire in breast cancer (BC) progression. The role of macrophages in early breast carcinogenesis is less understood. Macrophages adopt microenvironment-driven pro- or anti-inflammatory phenotypes. We evaluated macrophagic subsets in benign breast stroma to evaluate their association with future or concurrent BC. Methods: With institutional approval, FFPE tissues were identified from age-matched subjects, [with index benign breast biopsies and (a subset) interval-matched second benign tissues taken at the time of one of three events: ER+ BC, ER- BC or no cancer event (Controls) (index N=32, 26 and 30 respectively)]. Tissues were immunostained (CD68 or CD163) and stromal CD68+ and CD163+ macrophagic cell densities (MCD) (cells/mm2 stromal fibroadipose area) were digitally quantified. Statistical analysis was performed using Wilcoxon signed-rank tests for univariate analysis and conditional logistic regression models to adjust for BMI. Results: In the index age-matched biopsies (median age 54y), within subjects in all groups, the median CD163+ MCD (54.5; range: 13.9-371.3) was significantly higher than CD68+ MCD (19.6, range: 3.5-78.5) but the two were strongly correlated (r = 0.76); neither correlated with BMI (r&lt; 0.1 each). Across the groups, index benign biopsies of ER- BC had significantly lower CD68+ MCD and CD163+ MCD compared to ER+ BC (CD163: median 47.2 vs. 57.8, p=0.004; and median CD68+ MCD: 15.8 vs. 29.7, p=0.03); and ER- BC had significantly lower CD163+ MCD density than Controls (p=0.02), (all by univariate analysis and after adjusting for BMI). Comparing the index biopsy and second biopsy/excision within subjects, all showed significant increases in CD68+ MCD (median increase 27.9 for ER- BC, 16.1 for ER+ BC, and 17.8 for controls, p&lt;0.01), and only ER-BC showed an increase in CD163+ MCD (median increase 26.6, p=0.01). Thus, in the second biopsies/excisions, the CD163+ MCD and CD68+ MCD were similar across groups (median CD163+ MCD: 69.3, 75.4, and 59.3 and median CD68+ MCD: 47.4, 45.2, and 36.0 for ER+ BC, ER- BC, and controls); however, all groups had decreased CD163+:CD68+ MCD ratios compared to the index biopsies. Conclusion: Benign breast tissues have higher stromal CD163+ MCD than CD68+ MCD irrespective of future BC events and independent of BMI. However, between index and second biopsies/excisions, the stromal CD68+ MCD differentially increased in all groups, lowering CD163+:CD68+ MCD ratios, and likely shifting pro- and anti-inflammatory stromal macrophage functionality. Across groups, ER- BC index biopsies had significantly lower stromal MCD of both types compared to ER+ BC, and were the only group to increase stromal CD163+ MCD between index biopsy and cancer event. These data suggest that ER+ and ER- BC may have different pre-carcinogenic stromal inflammatory microenvironments. Citation Format: Jodi M. Carter, Ethan P. Heinzen, Tanya L. Hoskin, Stacey J. Winham, Derek C. Radisky, Daniel W. Visscher, Amy C. Degnim. Differential stromal macrophagic profiles precede ER+ and ER- breast carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2705.

1501 Background: Despite BC risk reduction of 50-65% by preventive endocrine therapy (ET), very few at-risk women choose to take them. A woman’s perceived BC risk correlates with uptake of ET. A PRS comprised of 77 BC genetic susceptibility loci (Single Nucleotide Polymorphisms (SNP) improves the accuracy of risk prediction for BC. We examined the impact of the addition of individualized PRS BC risk prediction to standard risk calculator estimates on intent to take BC prevention medication. Methods: Eligible women had ≥5% 10 yr BC Tyrer-Cuzick risk (IBIS) or 5 year Gail score ≥3%, with no history of BC or hereditary BC syndrome. Standard BC risk estimates (IBIS or Gail) were incorporated into the counselling on BC preventive ET. A self-reported questionnaire at baseline quantified intention to take ET and explored factors associated with this decision. Blood samples were obtained and genotyped for 77 SNPs, individualized PRS were calculated then incorporated into IBIS and Gail predictions for 5 yr, 10 yr, & lifetime BC risk. At a second visit, PRS risk & prevention recommendations were revisited. Post visit questionnaires assessed change in intent to take ET. Multivariable linear regression was performed to assess impact of baseline variables on change in intent to take medication. Results: From 2016 to 2017, 151 women in Canada & USA were enrolled, median age: 56.1 (range 36-76.4), 35.6% were premenopausal, 98.7% were Caucasian. Median 5yr, 10yr, & lifetime IBIS risk estimates were 3.8% (2.0-11.5), 7.9% (5.0-23.1), and 25.3% (5.5 to 92.2). PRS increased BC risk estimates in 84 (55.6%) and reduced BC risk estimates in 67 (44%) women. After PRS risk counselling, intention to take ET significantly changed (p<0.001): 41.9% of those with increased PRS were more inclined, and 46.7% of women with decreased PRS were less inclined to take ET. On multivariable regression, increase in PRS (p<0.0001) and less concern about ET side effects (p<0.0001) were associated with greater intent to take ET. Conclusions: In high risk women, PRS significantly changed BC risk estimates & intent to take preventive ET. Further assessments of the impact of PRS scores on compliance with ET are warranted. Clinical trial information: NCT02517593.