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Background Chronic inflammatory responses initiated by lymphatic injury play a key role in the pathophysiology of secondary lymphedema. However, it is unclear if baseline inflammation or ethnic/racial variability in inflammatory responses increase lymphedema risk. Crown-like structures of the breast (CLS-B), consisting of macrophages engulfing necrotic adipocytes, are a marker of systemic inflammation and have been implicated in the pathogenesis of breast cancer, but their role in lymphedema development is unknown. Here we determine whether baseline differences in inflammation, characterized by the presence of CLS-B, contributed to lymphedema risk in a diverse cohort of patients treated with ALND. Methods Patients ≥ 18 years undergoing ALND were enrolled in a prospective lymphedema screening study. Body mass index (BMI) and volumetric arm measurements (perometer) were performed at baseline, postoperatively, and every 6 months. Breast tissue obtained at definitive surgery was assessed for CLS-B with CD-68 IHC stain in non-tumor breast tissue. Inflammation severity was determined by number of CLS-B/cm2, with the median used to differentiate between mild and severe inflammation. Lymphedema was defined as a relative arm volume change of ≥10%. Lymphedema incidence was assessed using competing risk analysis and compared between patients with and without CLS-B. Uni- and multivariable analysis was performed to identify factors associated with lymphedema development. Results Between 11/2016-03/2020, 304 ALND patients were enrolled; 281 had at least 6 months of follow-up and were included in the study. Eleven percent self-identified as Asian, 20% Black, 6% Hispanic, and 60% White. Median age was 48 years; median BMI was 26.3 kg/m2, with higher BMI observed in Black and Hispanic women compared to Asian and White women (p < 0.001). Overall, 54% had CLS-B, with severe inflammation (> 0.4 CLS-B/cm2) identified in 71 (25%) patients. CLS-B presence correlated with BMI (36% [BMI < 25], 63% [BMI 25-30], 70% [BMI > 30], p < 0.001) and varied across racial/ethnic groups, with a higher prevalence in Black and Hispanic women (68% [Black], 69% [Hispanic] vs 59% [Asian], 46% [White], p = 0.03) (Table). Inflammation severity did not differ by race/ethnicity (p = 0.11). At 2.1 years median follow-up (IQR 1.6-3.1), 66 women developed lymphedema, with a 2-year lymphedema rate of 21.3% (95% CI 16.4-26.8). Lymphedema incidence was higher among Black and Hispanic women, compared to Asian and White women (2-year rate: 33.8% [Black], 31% [Hispanic], 17.4% [Asian], 18.2% [White], p = 0.002), and was higher among women with CLS-B (2-year rate: 28.2% [CLS-B] vs 12.9% [no CLS-B], p = 0.02). On multivariable analysis, Black race (White [referent]: HR 2.85, 95% CI 1.4-5.8; p = 0.03), receipt of NAC (upfront surgery [referent]: HR 2.46, 95% CI 1.04-5.8, p = 0.04) and older age (HR 1.03, 95% CI 1.01-1.06 per 1-year increase; p = 0.009) were independently associated with lymphedema development, while CLS-B was not (HR 1.37, 95% CI 0.81-2.34, p = 0.2). Conclusions In a prospective cohort of patients treated with ALND, Black race, receipt of NAC, and increasing age, but not CLS-B, were independently associated with lymphedema risk. However, the higher CLS-B prevalence in Black women suggests that they may have a propensity for increased inflammation, which may in part be contributing to the higher lymphedema risk observed, but is likely not the only inflammatory mechanism that modulates risk. Table. Clinical characteristics of study cohort stratified by the presence of CLS-B Citation Format: Andrea V. Barrio, Giacomo Montagna, Varadan Sevilimedu, Ethan Gomez, Dilip Giri, Babak Mehrara, Monica Morrow. Does Breast Inflammation Contribute to Lymphedema Risk in Patients Treated with Axillary Lymph Node Dissection? [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-08-09.

Supplementary Data from Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

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Human Energy Responsive Methylation Changes in Normal Breast Tissue.

Supplementary Figure 1 Legend.

Mouse Mapping Statistics, n=3/group.

IPA Biofunction Analysis. Significant disease or biofunction annotation implicated by IPA software.

Regulation of pro-growth and inflammatory gene expression.

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339–48. ©2016 AACR.

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Progression in digital pathology has yielded new opportunities for a remote work environment. We evaluated the utility of digital review of breast cancer immunohistochemical prognostic markers (IHC) using whole slide images (WSI) from formalin fixed paraffin embedded (FFPE) cytology cell block specimens (CB) using three different scanners.CB from 20 patients with breast cancer diagnosis and available IHC were included. Glass slides including 20 Hematoxylin and eosin (H&E), 20 Estrogen Receptor (ER), 20 Progesterone Receptor (PR), 16 Androgen Receptor (AR), and 20 Human Epidermal Growth Factor Receptor 2 (HER2) were scanned on 3 different scanners. Four breast pathologists reviewed the WSI and recorded their semi-quantitative scoring for each marker. Kappa concordance was defined as complete agreement between glass/digital pairs. Discordances between microscopic and digital reads were classified as a major when a clinically relevant change was seen. Minor discordances were defined as differences in scoring percentages/staining pattern that would not have resulted in a clinical implication. Scanner precision was tabulated according to the success rate of each scan on all three scanners.In total, we had 228 paired glass/digital IHC reads on all 3 scanners. There was strong concordance kappa ≥0.85 for all pathologists when comparing paired microscopic/digital reads. Strong concordance (kappa ≥0.86) was also seen when comparing reads between scanners.Twenty-three percent of the WSI required rescanning due to barcode detection failures, 14% due to tissue detection failures, and 2% due to focus issues. Scanner 1 had the best average precision of 92%. HER2 IHC had the lowest intra-scanner precision (64%) among all stains.This study is the first to address the utility of WSI in breast cancer IHC in CB and to validate its reporting using 3 different scanners. Digital images are reliable for breast IHC assessment in CB and offer similar reproducibility to microscope reads.

Purpose: HER2-amplified breast cancer is sometimes clinically insensitive to HER2-targeted treatment with trastuzumab. Laboratory models of resistance have causally implicated changes in HER2 expression and activation of the phosphoinositide 3-kinase (PI3K)–AKT pathway. We conducted a prospective tissue acquisition study to determine if there is evidence for these lesions in metastatic tumors that have progressed on trastuzumab-containing therapy.Experimental Design: From 2/2007 to 11/2011, 63 patients with HER2-amplified breast cancer with recurrence of disease after adjuvant trastuzumab therapy or World Health Organization–defined progression of metastatic disease on a trastuzumab-containing regimen were prospectively enrolled and underwent tumor biopsy. Specimens were analyzed for activating mutations in PIK3CA and HER2 by Sequenom and analyzed for HER2 and PTEN status by immunohistochemistry.Results: In 53/60 cases (88%, 3 cases not evaluable for HER2), HER2 overexpression persisted in the metastatic tumor following trastuzumab exposure. Among the 7 cases lacking HER2 overexpression, repeat analysis of the pretreatment tumor failed to confirm HER2 overexpression in five cases. Among cases evaluable for PTEN (56) or PI3K mutation (45), absent or significantly diminished PTEN expression was noted in 33 (59%) and activating mutations in PIK3CA in 13 (29%). The combined rate of PTEN loss and PIK3CA mutation in the trastuzumab-refractory tumors was 71% compared with 44% (P = 0.007) in an unexposed cohort of 73 HER2-amplified tumors.Conclusions: In this series of prospectively collected trastuzumab-refractory human breast cancers, loss of HER2 overexpression was rare, whereas activation of the PI3K-AKT pathway through loss of PTEN or PIK3CA mutation was frequently observed. Clin Cancer Res; 18(24); 6784–91. ©2012 AACR.

PDF file - 226KB, Representative micrographs of DCIS and invasive breast cancers displaying PTEN expression (A, left) or loss (A, right), INPP4B expression (B, left) or loss (B right), pAKT expression lack of expression (C, left) and pAKT expression in neoplastic cells (C, right), pS6 lack of expression (D, left) and pS6 expression in neoplastic cells (D, right). Please note that in A and B, the expression of PTEN and INPP4B in stromal cells and/or normal breast epithelium was employed as an internal positive control in cases lacking PTEN and INPP4B expression in neoplastic cells, respectively.

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PDF file - 23KB, Characteristics of patients with pure high-grade DCIS and high-grade DCIS associated with IBC included in this study.

Purpose: To assess the prevalence of phosphoinositide 3-kinase (PI3K) pathway alterations in pure high-grade ductal carcinoma in situ (DCIS) and DCIS associated with invasive breast cancer (IBC), and to determine whether DCIS and adjacent IBCs harbor distinct PI3K pathway aberrations.Experimental Design: Eighty-nine cases of pure high-grade DCIS and 119 cases of high-grade DCIS associated with IBC were characterized according to estrogen receptor (ER) and HER2 status, subjected to immunohistochemical analysis of PTEN, INPP4B, phosphorylated (p)AKT and pS6 expression, and to microdissection followed by Sequenom genotyping of PIK3CA and AKT1 hotspot mutations.Results: Alterations affecting the PI3K pathway were found in a subset of pure DCIS and DCIS adjacent to IBC. A subtype-matched comparison of pure DCIS and DCIS adjacent to IBC revealed that PIK3CA hotspot mutations and pAKT expression were significantly more prevalent in ER-positive/HER2-negative DCIS adjacent to IBC (P values, 0.005 and 0.043, respectively), and that in ER-negative/HER2-positive cases INPP4B loss of expression was more frequently observed in pure DCIS (a P value of 0.013). No differences in the parameters analyzed were observed in a pairwise comparison of the in situ and invasive components of cases of DCIS and adjacent IBC. Analysis of the PIK3CA-mutant allelic frequencies in DCIS and synchronous IBC revealed cases in which PIK3CA mutations were either restricted to the DCIS or to the invasive components.Conclusion: Molecular aberrations affecting the PI3K pathway may play a role in the progression from high-grade DCIS to IBC in a subset of cases (e.g., a subgroup of ER-positive/HER2-negative lesions). Clin Cancer Res; 20(9); 2326–37. ©2014 AACR.

PDF file - 1392K, PTEN IHC changes from pre-trastuzumab to post-trastuzumab samples. Depicted are photomicrographs of PTEN change from reduced to loss of expression: a) PTEN expression by IHC in the primary breast invasive carcinoma with reduced nuclear and cytoplasmic staining as compared to the adjacent normal stroma (200X); b) HE staining of the primary lesion (200X); c) PTEN complete loss of expression in the secondary invasive carcinoma with absence of any staining in the lesion as compared to the adjacent normal stroma (200X); d) HE staining of the secondary lesion (200X)

PDF file - 109K, Supplementary Table 1. Changes in HER2 status Supplementary Table 2. Changes in PTEN status Supplementary Table 3. Changes in PIK3CA status Supplementary Table 4. PI3K mutation and PTEN loss are not mutually exclusive Supplementary Table 5. ER/PR status among PI3K activated tumors

PDF file - 38K, Data flow diagram of the secondary/progressive lesions

PDF file - 39KB, PI3K pathway alterations in pure high-grade DCIS, high-grade DCIS components adjacent to IBC, and in synchronous IBC components.

PDF file - 58KB, PIK3CA mutations and mutation frequencies in pure high-grade DCIS and high-grade DCIS associated with IBC.

Supplementary Figure 3 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Invasive breast cancer has a high risk of recurrence to incurable disease and needs improved prognostic and therapeutic tools. Our work combines clinical and molecular analyses to show that the transcriptional repressor HBP1 may be a new target for invasive breast cancer. Previous work indicated that HBP1 regulated proliferation and senescence and inhibited Wnt signaling. Two of these functions have been associated with invasive breast cancer. In 76 breast tumors, we identified 10 HBP1 mutations/variants that were associated with fully invasive breast cancer. In a separate analysis, we found that a subset of invasive breast cancer specimens also had reduced HBP1 mRNA levels. These clinical correlations suggested that mutation or reduction of HBP1 occurs in invasive breast cancer and that HBP1 might regulate the proliferation and invasiveness of this breast cancer type. Analysis of the HBP1 mutants showed they were functionally defective for suppressing Wnt signaling. To test the consequences of reduced HBP1 levels, we used RNA interference to knock down HBP1 and observed increased Wnt signaling, tumorigenic proliferation, and invasiveness in cell and animal breast cancer models. Lastly, statistical analysis of a breast cancer patient database linked reduced HBP1 expression to breast cancer recurrence. In considering two-gene criteria for relapse potential, reduced expression of HBP1 and SFRP1, which is another Wnt inhibitor that was recently linked to invasive breast cancer, strikingly correlated with recurrence. Together, these data indicate that HBP1 may be a molecularly and clinically relevant regulator of breast cancer transitions that eventually lead to poor prognosis. [Cancer Res 2007;67(13):6136–45]

Supplementary Figure 2 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Supplementary Figure 1 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Supplementary Table 1 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Obesity is a risk factor for the development of post-menopausal breast cancer. Breast white adipose tissue (WAT) inflammation, which is commonly found in women with excess body fat, is also associated with increased breast cancer risk. Both local and systemic effects are probably important for explaining the link between excess body fat, adipose inflammation and breast cancer. The first goal of this cross-sectional study of 196 women was to carry out transcriptome profiling to define the molecular changes that occur in the breast related to excess body fat and WAT inflammation. A second objective was to determine if commonly measured blood biomarkers of risk and prognosis reflect molecular changes in the breast. Breast WAT inflammation was assessed by immunohistochemistry. Bulk RNA-sequencing was carried out to assess gene expression in non-tumorous breast. Obesity and WAT inflammation were associated with a large number of differentially expressed genes and changes in multiple pathways linked to the development and progression of breast cancer. Altered pathways included inflammatory response, complement, KRAS signaling, tumor necrosis factor α signaling via NFkB, interleukin (IL)6-JAK-STAT3 signaling, epithelial mesenchymal transition, angiogenesis, interferon γ response and transforming growth factor (TGF)-β signaling. Increased expression of several drug targets such as aromatase, TGF-β1, IDO-1 and PD-1 were observed. Levels of various blood biomarkers including high sensitivity C-reactive protein, IL6, leptin, adiponectin, triglycerides, high-density lipoprotein cholesterol and insulin were altered and correlated with molecular changes in the breast. Collectively, this study helps to explain both the link between obesity and breast cancer and the utility of blood biomarkers for determining risk and prognosis.

Objective Broad adoption of digital pathology (DP) is still lacking, and examples for DP connecting diagnostic, research, and educational use cases are missing. We blueprint a holistic DP solution at a large academic medical center ubiquitously integrated into clinical workflows; researchapplications including molecular, genetic, and tissue databases; and educational processes. Materials and Methods We built a vendor-agnostic, integrated viewer for reviewing, annotating, sharing, and quality assurance of digital slides in a clinical or research context. It is the first homegrown viewer cleared by New York State provisional approval in 2020 for primary diagnosis and remote sign-out during the COVID-19 (coronavirus disease 2019) pandemic. We further introduce an interconnected Honest Broker for BioInformatics Technology (HoBBIT) to systematically compile and share large-scale DP research datasets including anonymized images, redacted pathology reports, and clinical data of patients with consent. Results The solution has been operationally used over 3 years by 926 pathologists and researchers evaluating 288 903 digital slides. A total of 51% of these were reviewed within 1 month after scanning. Seamless integration of the viewer into 4 hospital systems clearly increases the adoption of DP. HoBBIT directly impacts the translation of knowledge in pathology into effective new health measures, including artificial intelligence–driven detection models for prostate cancer, basal cell carcinoma, and breast cancer metastases, developed and validated on thousands of cases. Conclusions We highlight major challenges and lessons learned when going digital to provide orientation for other pathologists. Building interconnected solutions will not only increase adoption of DP, but also facilitate next-generation computational pathology at scale for enhanced cancer research.

Obesity is associated with an increased risk of breast cancer in post-menopausal women and decreased risk in pre-menopausal women. Conversely, in BRCA1/2 mutation carriers, pre-menopausal obesity is associated with early-onset breast cancer. Here we show that obese, pre-menopausal BRCA1/2 mutation carriers have increased levels of aromatase and inflammation in the breast, as occurs in post-menopausal women. In a prospective cohort study of 141 women with germline BRCA1 (n = 74) or BRCA2 (n = 67) mutations, leptin, and aromatase expression were higher in the breast tissue of obese versus lean individuals (P P P BRCA1/2 mutation carriers.

Context.— The Ventana programmed death ligand-1 (PD-L1) SP142 immunohistochemical assay (IHC) is approved by the US Food and Drug Administration as the companion diagnostic assay to identify patients with locally advanced or metastatic triple-negative breast cancer for immunotherapy with atezolizumab, a monoclonal antibody targeting PD-L1. Objective.— To determine interobserver variability in PD-L1 SP142 IHC interpretation in invasive breast carcinoma. Design.— The pathology database was interrogated for all patients diagnosed with primary invasive, locally recurrent, or metastatic breast carcinoma on which PD-L1 SP142 IHC was performed from November 2018 to June 2019 at our institution. A subset of cases was selected using a computerized random-number generator. PD-L1 IHC was evaluated in stromal tumor-infiltrating immune cells using the IMpassion130 trial criteria, with positive cases defined as immunoreactivity in immune cells in 1% or more of the tumor area. IHC was interpreted on whole slide images by staff pathologists with breast pathology expertise. Interobserver variability was calculated using unweighted κ. Results.— A total of 79 cases were assessed by 8 pathologists. Interobserver agreement was substantial (κ = 0.727). There was complete agreement among all 8 pathologists in 62% (49 of 79) of cases, 7 pathologists or more in 84% (66 of 79) of cases, and 6 pathologists or more in 92% (73 of 79) of cases. In 4% (3 of 79) of cases, all of which were small biopsies, pathologists' interpretations were evenly split between scores of positive and negative. Conclusions.— The findings show substantial agreement in PD-L1 SP142 IHC assessment of breast carcinoma cases among 8 pathologists at a single institution. Further study is warranted to define the basis for discrepant results.

Metaplastic breast carcinoma (MBC) is a rare, aggressive subtype of breast cancer associated with poorer overall survival than other triple-negative breast cancers. This study sought to compare survival outcomes among histologic subtypes of MBC with those of non-metaplastic triple-negative breast cancer. Clinicopathologic and treatment data for all patients with non-metastatic, pure MBC undergoing surgery from 1995 to 2017 and for a large cohort of patients with other types of triple-negative breast cancer during that period were collected from an institutional database. The MBC tumors were classified as having squamous, spindle, heterologous mesenchymal, or mixed histology. Survival outcomes were compared using the Kaplan-Meier method. Of 132 MBC patients, those with heterologous mesenchymal MBC (n = 45) had the best 5-year overall and breast cancer-specific survival (BCSS, 88%; 95% confidence interval [CI], 0.78–0.99), whereas those with squamous MBC had the worst survival (BCSS, 56%; 95% CI, 0.32–0.79). Overall survival, BCSS, and recurrence-free survival were worse for the patients with MBC than for the patients who had non-MBC triple-negative breast cancer, with a clinicopathologically adjusted recurrence hazard ratio of 2.4 (95% CI, 1.6–3.3; p

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2 R172 hotspot mutations or TET2 mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2 R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2 R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2 R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2 R172 hotspot mutations, which co-occurred with PIK3CA hotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2 R172 mutations, however PIK3CA hotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n = 9/10) and core needle biopsy specimens (n = 5), whereas the remaining papillary neoplasms (n = 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2 R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.

Objectives We sought to determine the safety and efficacy of the oral androgen receptor antagonist enzalutamide in patients with previously treated, recurrent, AR-positive (AR+) ovarian cancer. Methods This was a single-institution phase II study of patients with AR+ ovarian cancer with measurable disease with 1–3 prior lines of chemotherapy; patients were screened for enrollment from 11/2013–7/2018. Following consent, archival tissue was evaluated for AR+. Enrolled patients received daily enzalutamide 160 mg until progression of disease or treatment discontinuation. Adverse events were graded by CTCAE v4.0. Co-primary endpoints were 6-month progression-free survival (PFS6) and overall response rate (ORR) by RECIST 1.1 criteria. Results During the study period, 160 patients were screened and 59 (45 high-grade serous [HGS] and 14 low-grade serous [LGS]) consented to treatment on study. There was 1 confirmed and 1 unconfirmed partial response. The ORR was 1.7% (90% CI: 0.2–100%). The overall PFS6 rate (as binary) was 22% (90% CI: 15.1–100%). The 6-month PFS rate (as time to event) was 19.8% for HGS patients (90% CI: 12.7–100%) and 38.5% (90% CI: 21.7%–100%) for LGS patients. Grade 3 toxicities occurred in 6 patients (one toxicity (Grade 3 rash) was considered a dose-limiting toxicity). One patient died of cardiac arrest after 42 days on treatment of a cardiac arrest not attributed to study drug. Conclusions The study met its primary endpoint, with a PFS6 rate of 22% (n = 13); however, the overall response rate was low. Enzalutamide was well tolerated and may be a potential treatment option in select patients.

Toxoplasmosis is a rare but potentially severe complication after allogeneic hematopoietic cell transplantation. Toxoplasma gondii-associated cardiac involvement can cause myocarditis, pericarditis, arrhythmias, and congestive heart failure. Most cases with cardiac toxoplasmosis following BMT have been fatal and diagnosed at autopsy. We present an unfortunate case of sudden onset congestive heart failure symptoms and delayed post-transplant Toxoplasma PCR testing that ultimately led to the diagnosis of cardiac toxoplasmosis on autopsy in our patient.

Lobular carcinoma in situ (LCIS) is a risk factor for breast cancer, but the effect of LCIS found in association with ductal carcinoma in situ (DCIS) is unknown. In this study, we compared contralateral breast cancer (CBC) and ipsilateral breast tumor recurrence (IBTR) rates among women with DCIS with or without synchronous ipsilateral LCIS treated with breast-conserving surgery (BCS). DCIS patients undergoing BCS from 2000 to 2011 with a contralateral breast at risk were stratified by the presence or absence of synchronous ipsilateral LCIS with the index DCIS (DCIS + LCIS vs. DCIS). Those with contralateral, bilateral, or prior ipsilateral LCIS were excluded. Associations of patient, tumor, and treatment factors with CBC and IBTR were evaluated. Of 1888 patients identified, 1475 (78%) had DCIS and 413 (22%) had DCIS + LCIS. At median follow-up of 7.2 (range 0–17) years, 307 patients had a subsequent first breast event; 207 IBTR and 100 CBC. The 10-year cumulative incidence of IBTR was similar in both groups: 15.0% vs. 14.2% (log-rank, p = 0.8) for DCIS + LCIS vs. DCIS, respectively. The 10-year cumulative incidence of CBC was greater in the DCIS + LCIS group: 10.9% vs. 6.1% for DCIS (log-rank, p

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: LCIS is considered a risk factor for bilateral breast cancer, but the effect of LCIS diagnosed concurrently with DCIS is not known. We sought to compare CBC and ipsilateral breast tumor recurrence (IBTR) rates in women with DCIS with and without synchronous LCIS treated with breast conserving surgery (BCS). Methods: A prospectively maintained database of DCIS patients undergoing BCS from 2000-2011 was used to identify women with a contralateral breast at risk. Patients with synchronous ipsilateral LCIS found at core needle biopsy or surgical excision were included in the “DCIS + LCIS” group; those with contralateral or bilateral LCIS were excluded. Associations of patient, tumor, and treatment factors with CBC and IBTR were evaluated using logistic regression. Results: Of the 1888 patients identified, 1475 (78%) had DCIS only and 413 (22%) had DCIS with synchronous LCIS. Median follow-up was 7.5 years (range 0-17 years). 305 patients had a subsequent breast event; 216 IBTR and 89 CBC. The 5 and 10-year cumulative incidence of IBTR was similar in both groups: 6.3% and 14.4% for DCIS only, compared with 5.9% and 14.0% for DCIS + LCIS (p = 0.94), respectively. The 5 and 10-year cumulative incidence of CBC was significantly greater in the DCIS + LCIS group: 5.7% and 10.0%, compared with 2.4% and 5.0% for DCIS only (p < 0.001). Table 1 summarizes uni-and multi-variable analyses of risk factors associated with CBC and IBTR among women with DCIS treated with BCS. After adjustment for other factors, CBC risk was more than 2-fold higher in the DCIS + LCIS group compared with the DCIS only group (HR 2.37, 95% CI 1.54-3.65, p < 0.001). There was no difference in IBTR risk based on presence of synchronous LCIS. Younger age and receipt of endocrine therapy were significantly associated with decreased risk of CBC. Table 1:Risk factors associated with CBC and IBTR in patients with DCIS treated with BCS Univariate Analysis Multivariate Analysis Hazard Ratio (95% CI) Hazard Ratio (95% CI) CBC IBTR CBC IBTRAge (continuous)1.02 (1.01-1.04) *0.99 (0.98-1.0)1.02 (1.0-1.04) *0.99 (0.97-1.0) *Presentation Radiologic1111Clinical0.36 (0.11-1.14)1.56 (1.05-2.3) *0.41 (0.13-1.31)1.67 (1.12-2.47) *Family history Yes1.32 (0.87-2.01)1.0 (0.76-1.32)1.33 (0.87-2.03)1.03 (0.78-1.35)Grade Low1111Intermediate/high1.83 (1.0-3.37)1.32 (0.93-1.88)1.74 (0.92-3.29)1.62 (1.11-2.35) *Radiation Yes1.16 (0.75-1.8)0.7 (0.54-0.92) *1.14 (0.71-1.82)0.64 (0.48-0.86) *Endocrine Therapy Yes0.43 (0.23-0.81) *0.51 (0.35-0.74) *0.42 (0.22-0.8) *0.53 (0.36-0.77) *DCIS group DCIS only1111DCIS + LCIS2.28 (1.49-3.5) *0.99 (0.71-1.36)2.37 (1.54-3.65) *0.99 (0.71-1.37)* p < 0.05 Conclusions: LCIS diagnosed concurrently with DCIS is not associated with IBTR, but increases the risk of CBC two-fold. Endocrine therapy should be considered both for the index DCIS and for prevention of subsequent CBC. Citation Format: Miller ME, Muhsen S, Zabor EC, Flynn J, Olcese C, Giri D, Van Zee KJ, Pilewskie M. Risk of contralateral breast cancer (CBC) in women with ductal carcinoma in situ (DCIS) with and without and synchronous lobular carcinoma in situ (LCIS) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-18-01.

Purpose: Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers. Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods. Results: WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases. Conclusions: Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

Diffusion-weighted imaging is a non-invasive functional imaging modality for breast tumor characterization through apparent diffusion coefficients. Yet, it has so far been unable to intuitively inform on tissue microstructure. In this IRB-approved prospective study, we applied novel multidimensional diffusion (MDD) encoding across 16 patients with suspected breast cancer to evaluate its potential for tissue characterization in the clinical setting. Data acquired via custom MDD sequences was processed using an algorithm estimating non-parametric diffusion tensor distributions. The statistical descriptors of these distributions allow us to quantify tissue composition in terms of metrics informing on cell densities, shapes, and orientations. Additionally, signal fractions from specific cell types, such as elongated cells (bin1), isotropic cells (bin2), and free water (bin3), were teased apart. Histogram analysis in cancers and healthy breast tissue showed that cancers exhibited lower mean values of “size” (1.43 ± 0.54 × 10−3 mm2/s) and higher mean values of “shape” (0.47 ± 0.15) corresponding to bin1, while FGT (fibroglandular breast tissue) presented higher mean values of “size” (2.33 ± 0.22 × 10−3 mm2/s) and lower mean values of “shape” (0.27 ± 0.11) corresponding to bin3 (p &lt, 0.001). Invasive carcinomas showed significant differences in mean signal fractions from bin1 (0.64 ± 0.13 vs. 0.4 ± 0.25) and bin3 (0.18 ± 0.08 vs. 0.42 ± 0.21) compared to ductal carcinomas in situ (DCIS) and invasive carcinomas with associated DCIS (p = 0.03). MDD enabled qualitative and quantitative evaluation of the composition of breast cancers and healthy glands.

Obesity is a risk factor for the development of hormone receptor-positive breast cancer in post-menopausal women. Estrogen synthesis is catalyzed by aromatase. Recently, we identified an obesity-inflammation-aromatase axis in mouse models and women. In mouse models of obesity, inflammatory foci characterized by crown-like structures (CLS) consisting of dead adipocytes encircled by macrophages were found in the mammary gland (MG). CLS of the breast were found in most overweight and obese women. CLS were associated with adipocyte hypertrophy, activation of NF-κB, elevated levels of proinflammatory mediators and aromatase, and increased expression of the progesterone receptor (PR). Collectively, these findings provide a plausible explanation for the link between obesity, chronic inflammation, and post-menopausal breast cancer. Here we investigated whether caloric restriction (CR) reversed the inflammatory state and related molecular changes in the MG of obese mice. Obese ovariectomized C57BL/6J mice were subjected to 30% CR for 7 or 14 weeks. Findings in CR mice were compared with results in mice fed a high fat diet ad libitum or with control mice fed a low fat diet. CR was associated with more than a 75% decrease in mammary CLS/cm2. Reduced histological inflammation following CR was associated with decreased adipocyte diameter and MCP-1 levels, reduced NF-κB binding activity, and normalization of levels of proinflammatory mediators, aromatase and PR. In summary, obesity-related inflammation of the MG and elevated aromatase and PR levels were reversed with CR. Our results provide a rationale for determining whether weight loss can reverse breast inflammation associated with obesity in women.

Excess body fat and sedentary behavior are associated with increased breast cancer risk and mortality, including in normal weight women. To investigate underlying mechanisms, we examined whether adiposity and exercise impact the breast microenvironment (e.g., inflammation and aromatase expression) and circulating metabo-inflammatory factors. In a cross-sectional cohort study, breast white adipose tissue (WAT) and blood were collected from 100 women undergoing mastectomy for breast cancer risk reduction or treatment. Self-reported exercise behavior, body composition measured by dual-energy x-ray absorptiometry (DXA), and waist:hip ratio were obtained prior to surgery. Breast WAT inflammation (B-WATi) was assessed by IHC and aromatase expression was assessed by quantitative PCR. Metabolic and inflammatory blood biomarkers that are predictive of breast cancer risk and progression were measured. B-WATi was present in 56 of 100 patients and was associated with older age, elevated BMI, postmenopausal status, decreased exercise, hypertension and dyslipidemia (Ps < 0.001). Total body fat and trunk fat correlated with B-WATi and breast aromatase levels (Ps < 0.001). Circulating C-reactive protein, IL6, insulin, and leptin positively correlated with body fat and breast aromatase levels, while negative correlations were observed for adiponectin and sex hormone binding globulin (P < 0.001). Inverse relationships were observed with exercise (Ps < 0.05). In a subgroup of 39 women with normal BMI, body fat levels positively correlated with B-WATi and aromatase expression (Ps < 0.05). In conclusion, elevated body fat levels and decreased exercise are associated with protumorigenic micro- and host environments in normal, overweight, and obese individuals. These findings support the development of BMI-agnostic lifestyle interventions that target adiposity. Prevention Relevance: We report that individuals with high body fat and low exercise levels have breast inflammation, higher breast aromatase expression, and levels of circulating metabo-inflammatory factors that have been associated with increased breast cancer risk. These findings support interventions to lower adiposity, even among normal weight individuals, to prevent tumor growth.

Obesity is associated with an increased incidence of high-grade prostate cancer and poor prognosis for prostate cancer patients. Recently, we showed that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by crown-like structures (CLS) consisting of dead or dying adipocytes surrounded by macrophages, was associated with high-grade prostate cancer. It is possible, therefore, that agents that suppress periprostatic WAT inflammation will alter the development or progression of prostate cancer. Pioglitazone, a ligand of PPARγ, is used to treat diabetes and possesses anti-inflammatory properties. Here, our main objectives were to determine whether pioglitazone inhibited obesity-related periprostatic WAT inflammation in mice and then to elucidate the underlying mechanism. Treatment with pioglitazone reduced the density of CLS in periprostatic fat and suppressed levels of TNFα, TGFβ, and the chemokine monocyte chemoattractant protein-1 (MCP-1). Importantly, the ability of pioglitazone to suppress periprostatic WAT inflammation was abrogated in MCP-1 knockout mice. Pioglitazone caused dose-dependent induction of both adiponectin, an anti-inflammatory adipokine, and its receptor AdipoR2 in cultured 3T3-L1 cells and in periprostatic WAT of obese mice. Pioglitazone blocked TNFα-mediated induction of MCP-1 in 3T3-L1 cells, an effect that was attenuated when either adiponectin or AdipoR2 were silenced. Taken together, pioglitazone-mediated induction of adiponectin suppressed the elevation in MCP-1 levels, thereby attenuating obesity-related periprostatic WAT inflammation. These findings strengthen the rationale for future efforts to determine whether targeting the PPARγ–adiponectin–MCP-1 axis will decrease periprostatic adipose inflammation and thereby reduce the risk of high-grade prostate cancer or improve outcomes for men with prostate cancer. Cancer Prev Res; 11(4); 215–26. ©2017 AACR.

Background: Elevated body mass index (BMI) is associated with increased risk of estrogen receptor-positive postmenopausal breast cancer. Mechanistically, most individuals with elevated BMI have breast white adipose tissue inflammation (WATi) which confers increased breast cancer risk, particularly in those with existing benign breast disease. Individuals with WATi have elevated in-breast expression of aromatase and several systemic changes that increase breast cancer risk, including hyperinsulinemia and higher levels of C-reactive protein. However, women with normal BMI but high levels of body fat are also likely to harbor WATi and are at increased risk of postmenopausal breast cancer. The accuracy of BMI for assessing adiposity and predicting obesity-related disorders, including cancer, varies across race and ethnicity. Whether the association between BMI and WATi varies by race is unknown. Here we aimed to characterize relationships among breast WATi and clinicopathologic features in a racially diverse cohort undergoing mastectomy for breast cancer treatment. Methods: Non-tumorous breast tissue and fasting blood were collected from women undergoing mastectomy for breast cancer treatment or prevention at a single center serving a racially diverse patient population. Breast WATi was detected by the presence of crown-like structures in the breast (CLS-B), which are composed of a dead/dying adipocyte surrounded by CD68+ macrophages. Clinicopathologic data were abstracted from electronic medical records. Associations among categorical variables were examined using Fisher's exact test. Relationships between continuous variables were examined using the Spearman correlation. Results: As of May 18, 2018 62 patients have been accrued; median age 55 (range 32 to 84). Self-reported race distribution was: 36 (58%) Asian, 5 African American (8%), 20 (32%) Caucasian, and 1 (2%) unknown. Breast tissue has been analyzed for WATi in 60 cases thus far. Clinicopathologic features stratified by the presence or absence of breast WATi are presented in. Breast WAT inflammation was associated with obesity (P=0.02) and a trend to association was observed with dyslipidemia (P VariableBreast WATi Absent (n=25)Breast WATi Present (n=35)Age, years Median (range)51 (32 to 71)59 (36 to 80)BMI, kg/m2 Median (range)22.5 (18.1 to 35.3)28.0 (19.2 to 38.9)BMI Category Underweight1 (4%)0 (0%)Normal16 (64%)10 (29%)Overweight5 (20%)18 (51%)Obese3 (12%)7 (20%)Race, n (%) Asian15 (60%)19 (54%)African American2 (8%)3 (9%)Caucasian8 (32%)12 (34%)Unknown0 (0%)1 (3%)Menopausal Status, n (%) Pre10 (40%)12 (34%)Post15 (60%)23 (66%)Hypertension, n (%)7 (28%)16 (46%)Diabetes mellitus, n (%)0 (0%)5 (14%)Dyslipidemia, n (%)1 (4%)11 (31%) Conclusions: Breast adipose inflammation is associated with elevated BMI and possibly metabolic syndrome disorders in a racially diverse population. These findings are consistent with observations from predominantly Caucasian cohorts. Race-specific characteristics will also be examined. Study accrual is ongoing and updated results will be presented. Citation Format: Iyengar NM, Siegel B, Malik M, Giri DD, Tsai J, Hughes M, Adam A, Williams S, Zhou XK, Rodgers W, Ginter P, Patel A, Yong F, Cherian A, August P, Dannenberg AJ. Obesity, adipose inflammation, and race in patients with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-05.

Adipose tissue inflammation is associated with obesity comorbidities. Reducing such inflammation may ameliorate these comorbidities. n-3 fatty acids have been reported to have anti-inflammatory properties in obesity, which may modulate this inflammatory state. In the current study a 1 gram per day oral supplement of the n-3 fatty acid docosahexaenoic acid (DHA) was administered for 12 weeks to 10 grade 1-2 obese postmenopausal women and markers of adipose tissue and systemic inflammation measured and compared before and after supplementation. DHA administration resulted in approximately a doubling of plasma and red cell phospholipid and adipose tissue DHA content but no change in systemic markers of inflammation, such as circulating C-reactive protein (CRP) or interleukins (IL) 6, 8 and 10 (IL-6, IL-8, IL-10). DHA supplementation did not alter the adipose tissue marker of inflammation crown-like structure density nor did it affect any gene expression pathways, including anti-inflammatory, hypoxic and lipid metabolism pathways. The obese postmenopausal women studied were otherwise healthy, which leads us to suggest that in such women DHA supplementation is not an effective means for reducing adipose tissue or systemic inflammation. Further testing is warranted to determine if n-3 fatty acids may ameliorate inflammation in other, perhaps less healthy, populations of obese individuals.

Purpose To measure the apparent diffusion coefficient (ADC) values in estrogen receptor-positive (ER+) and axillary lymph node-negative (LN–) invasive breast cancer and investigate the correlation of ADC with Oncotype Dx test recurrence scores (ODxRS). Materials and Methods This was a Health Insurance Portability and Accountability Act (HIPAA)-compliant single-site retrospective study. Patients underwent preoperative 3.0T MRI scans with additional diffusion-weighted imaging sequential scans (b = 0, 600 and b = 0, 1000 s/mm2) from January 2011 to 2013. The study population included 31 ER+/LN– invasive breast cancers, which underwent ODxRS genomic testing. ADC600 and ADC1000 parametric maps were generated, and ADC values were calculated from a user-drawn region of interest. ODxRS predicts 10-year recurrence risk in individual patients: low (RS 30). All breast lesions, including subgroups of invasive ductal carcinoma (IDC) lesions and mass-only lesions were dichotomized by RS scores, low-risk versus intermediate/high-risk, and statistical analysis was performed using Mann–Whitney's test (statistical significance at P < 0.05) and receiver operating characteristic (ROC) curves. Multivariate analysis was also performed. Results Invasive breast cancers, when scored as low-risk by ODxRS, had significantly higher ADC values compared with intermediate/high-risk lesions for both ADC600 (P = 0.007) and ADC1000 (P = 0.008) mean values. This was true both when analyzing only mass-lesions (P = 0.03 and 0.01) or only IDCs (P = 0.001 and 0.009). Conclusion Preliminary findings suggest that lesion ADC values correlate with recurrence risk likelihood stratified using ODxRS. Hence, ADC is a potential surrogate biomarker for tumor aggressiveness. Level of Evidence: 3 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2017.

Background Obesity, a cause of subclinical inflammation, is associated with increased risk of high grade prostate cancer (PC) and poor outcomes. Whether inflammation occurs in periprostatic white adipose tissue (WAT), and contributes to the negative impact of obesity on PC aggressiveness, is unknown. Methods In a single-center, cross-sectional design, men with newly diagnosed PC undergoing radical prostatectomy were eligible for study participation. The primary objective was to examine the prevalence of periprostatic WAT inflammation defined by the presence of crown-like structures (CLS-P) as detected by CD68 immunohistochemistry. Secondary objectives were to explore the clinical and systemic correlates of periprostatic WAT inflammation. Tumor characteristics and host factors including BMI, adipocyte diameter, and circulating levels of lipids, adipokines, and other metabolic factors were measured. Wilcoxon rank-sum, Chi-square, or Fisher’s exact tests, and generalized linear regression were used to examine the association between WAT inflammation and tumor and host characteristics. Results Periprostatic fat was collected from 169 men (median age 62 years; median BMI 28.3). Periprostatic WAT inflammation was identified in 49.7% of patients and associated with higher BMI (P=0.02), larger adipocyte size (P=0.004), and Gleason grade groups IV/V tumors (P=0.02). The relationship between WAT inflammation and high Gleason grade remained significant after adjusting for BMI (P=0.04). WAT inflammation correlated with higher circulating levels of insulin, triglycerides, and leptin/adiponectin ratio, and lower high density lipoprotein cholesterol, compared to those without WAT inflammation (P’s