Spatial N-glycomics of the normal breast microenvironment reveals fucosylated and high-mannose N-glycan signatures related to BI-RADS density and ancestry.

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.

Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics.

Ductal carcinoma in situ (DCIS) is a heterogeneous breast disease that remains challenging to treat due to its unpredictable progression to invasive breast cancer (IBC). Contemporary literature has become increasingly focused on extracellular matrix (ECM) alterations with breast cancer progression. However, the spatial regulation of the ECM proteome in DCIS has yet to be investigated in relation to IBC. We hypothesized that DCIS and IBC present distinct ECM proteomes that could discriminate between these pathologies. Tissue sections of pure DCIS, mixed DCIS-IBC, or pure IBC (n = 22) with detailed pathological annotations were investigated by multiplexed spatial proteomics. Across tissues, 1,005 ECM peptides were detected in pathologically annotated regions and their surrounding extracellular microenvironments. A comparison of DCIS to IBC pathologies demonstrated 43 significantly altered ECM peptides. Notably, eight fibrillar collagen peptides could distinguish with high specificity and sensitivity between DCIS and IBC. Lesion-targeted proteomic imaging revealed heterogeneity of the ECM proteome surrounding individual DCIS lesions. Multiplexed spatial proteomics reported an invasive cancer field effect, in which DCIS lesions in closer proximity to IBC shared a more similar ECM profile to IBC than distal counterparts. Defining the ECM proteomic microenvironment provides novel molecular insights relating to DCIS and IBC.

MYC is sufficient to generate mid-life high-grade serous ovarian and uterine serous carcinomas in a p53-R270H mouse model.

Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment is understood. However, previous GEMMs of high-grade serous ovarian cancer (HGSOC) have had to utilize genetics rarely or never found in human HGSOC to yield ovarian cancer within the lifespan of a mouse. MYC, an oncogene, is amongst the most amplified genes in HGSOC, but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant negative mutant p53-R270H with a fallopian tube epithelium-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium (FTE). Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate the Myc and Trp53-R270H transgene was able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology directed repair mutations. Histological and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the fallopian tube epithelium.

The anti-proliferative effects of a frankincense extract in a window of opportunity phase ia clinical trial for patients with breast cancer.

PURPOSE: Boswellic acids, active components of frankincense, suppress tumor proliferation in vitro with a strong clinical trial safety profile in patients with inflammatory diseases. We performed a Phase Ia window of opportunity trial of Boswellia serrata (B. serrata) in patients with breast cancer to evaluate its biologic activity and safety. METHODS: Patients with invasive breast cancer were treated pre-operatively with B. Serrata (2400 mg/day PO) until the night before surgery for a median of 11 days (SD 6 days; range: 5-23 days). Paraffin-embedded sections from pretreatment diagnostic core biopsies and post-treatment surgical excisions were evaluated using a tunnel assay and immunohistochemistry staining with Ki-67 antibodies. A non-intervention retrospective control arm consisting of core and surgical tissue specimens from untreated patients was used to compare patients treated with B. Serrata. The change in proliferation and apoptosis between diagnostic core specimens and surgical specimens was compared between the control and treatment groups using a two-tailed paired t-test. RESULTS: Twenty-two patients were enrolled, of which 20 received treatment, and 18 had sufficient tissue for IHC. There was an increase in percent change in proliferation from core biopsy to surgical excision in the control group (n = 18) of 54.6 ± 21.4%. In the B. serrata-treated group there was a reduction in proliferation between core biopsy and excision (n = 18) of 13.8 ± 11.7%. This difference was statistically significant between the control and B. serrata-treated groups (p = 0.008). There was no difference in change in apoptosis. There were no serious adverse events related to the drug. CONCLUSION: Boswellia serrata inhibited breast cancer proliferation and was well-tolerated in a Phase Ia window of opportunity trial.

Angiolipoma an Uncommon Breast Mass in Men: A Case Series.

Angiolipomas are uncommon benign masses of the breast which are rarely described in the male breast. They do not have a typical mammographic appearance and can present with concerning features such as microcalcifications or irregular borders. Ultrasound is helpful in evaluating these masses most commonly appearing as oval, circumscribed, and hyperechoic. Clinical, radiological, and pathological information needs to be carefully evaluated as angiolipomas can be confused with malignant pathology. Three cases of angiolipomas of the male breast are reported in this study with mammographic, sonographic, and pathologic correlation.

Secreted frizzled related-protein 2 is prognostic for human pancreatic cancer patient survival and is associated with fibrosis.

Pancreatic adenocarcinoma (PDAC) is one of the deadliest cancers, with five-year survival rates of 9%. We hypothesized that secreted frizzled-related protein 2 (SFRP2) may influence stromal growth in pancreatic cancer, since it increases fibrosis and collagen production in non-neoplastic pathologies. We assessed SFRP2 value as a biomarker and assessed its function in PDAC. SFRP2 gene expression in patients with PDAC was analyzed using TCGA data. Disease free survival (DFS) was analyzed using Kaplan Meier test. The effect of KRAS inhibition on SFRP2 expression in PDAC cells was assessed. The associations of stromal content with SFPR2 mRNA and protein with fibrosis were analyzed. The role of SFRP2 in mesenchymal transformation was assessed by western blot in fibroblasts. Of all cancers in TCGA, SFRP2 levels were highest in PDAC, and higher in PDAC than normal tissues (n= 234, p= 0.0003). High SFRP2 levels correlated with decreased DFS (p= 0.0097). KRAS inhibition reduced SFRP2 levels. Spearman correlation was 0.81 between stromal RNA and SFRP2 in human PDAC, and 0.75 between fibrosis and SFRP2 levels in PDAC tumors. SFRP2-treated fibroblasts displayed mesenchymal characteristics. SFRP2 is prognostic for PDAC survival, regulated by KRAS, and associated with PDAC fibrosis.

Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland.

BACKGROUND: Advanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship. METHODS: To address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal-Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups. RESULTS: Histological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion. CONCLUSIONS: We observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

Non-enzymatic glycoxidation linked with nutrition enhances the tumorigenic capacity of prostate cancer epithelia through AGE mediated activation of RAGE in cancer associated fibroblasts.

The molecular implications of food consumption on cancer etiology are poorly defined. The rate of nutrition associated non-enzymatic glycoxidation, a reaction that occurs between reactive carbonyl groups on linear sugars and nucleophilic amino, lysyl and arginyl groups on fats and proteins, is rapidly increased by food cooking and manufacturing processes. In this study, we assign nutrition-associated glycoxidation with significant oncogenic potential, promoting prostate tumor growth, progression, and metastasis in vivo. Advanced glycation end products (AGEs) are the final irreversible product of non-enzymatic glycoxidation. Exogenous treatment of prostate tumor cells with a single AGE peptide replicated glycoxidation induced tumor growth in vivo. Mechanistically, receptor for AGE (RAGE) deficiency in the stroma inhibited AGE mediated tumor growth. Functionally, AGE treatment induced RAGE dimerization in activated fibroblasts which sustained and increased the migratory potential of tumor epithelial cells. These data identify a novel nutrition associated pathway that can promote a tissue microenvironment conducive for aggressive tumor growth. Targeted and/or interventional strategies aimed at reducing AGE bioavailability as a consequence of nutrition may be viewed as novel chemoprevention initiatives.

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.

Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge.

INTRODUCTION: Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples. AREAS COVERED: We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented. EXPERT OPINION: The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.

A case report of inverted Meckel's diverticulum.

Inverted Meckel's diverticulum is an entity often discovered incidentally or through a clinical evaluation for gastrointestinal bleeding. While rare, inverted Meckel's diverticulum should be considered in the evaluation of a patient presenting with gastrointestinal bleeding, intestinal obstruction, or intussusception. In this case, a 67-year-old female with a remote history of surgically treated breast cancer presents to an urgent care facility with weakness and fatigue. She was found to be anemic with hemoglobin of 4. Imaging revealed a blind-ending pouch in the mid to distal ileum consistent with an inverted Meckel's diverticulum. Inverted Meckel's diverticulum is identified on computerized tomography as an intraluminal, blind-ending structure in the mid to distal ileum. The possibility of a lead point should be investigated and surgical resection is indicated to prevent intestinal obstruction.

Peyronie disease: a clinicopathologic study of 71 cases with emphasis on histopathologic patterns and prevalent metaplastic ossification.

Peyronie disease (PD) is a benign, superficial fibromatosis involving the fascial structures of the penis, causing deformity, pain, and loss of function, for which there are few contemporary studies of the histopathology. We performed a multi-institutional review of 74 routine and consultation specimens submitted with clinical concern for PD. Of these, three non-PD lesions were identified and excluded (a myointimoma, a mammary-type myofibroblastoma, and fibrocalcific atherosclerosis). Of the 71 confirmed to be PD, the majority of patients were white (83%), with a median age of 55 years (range: 26-88). The dorsal aspect of the penis was the most common site involved (78%), followed by lateral (12%) and ventral (10%) aspects. The median degree of curvature was 70° (range: 20-360°). On review, three overall histologic patterns characterized the lesions resected: dense fibrotic plaque (61%), dense fibrotic plaque with focal or patchy metaplastic ossification (35%), and plaque composed predominantly of metaplastic ossification (4%). The fibrotic component was predominantly nodular (18%), hyalinized/lamellar (46%), or mixed (32%), excepting two cases consisting entirely of metaplastic bone. Chronic inflammation, when present, was most often focal and perivascular in distribution. In one case, an excision after collagenase treatment showed myxoid change and increased stromal cellularity. Overall, these findings define the range of PD histology, particularly emphasizing that the calcification noted clinically nearly always represents bona fide metaplastic ossification. Such context will be of value in evaluating specimens prospectively, in light of changing practices and the use of new technologies for treatment.

Zonal regulation of collagen-type proteins and posttranslational modifications in prostatic benign and cancer tissues by imaging mass spectrometry.

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.

#EBUSTwitter: Novel Use of Social Media for Conception, Coordination, and Completion of an International, Multicenter Pathology Study.

CONTEXT.­: Social media sites are increasingly used for education, networking, and rapid dissemination of medical information, but their utility for facilitating research has remained largely untapped. OBJECTIVE.­: To describe in detail our experience using a social media platform (Twitter) for the successful initiation, coordination, and completion of an international, multi-institution pathology research study. DESIGN.­: Following a tweet describing a hitherto-unreported biopsy-related histologic finding in a mediastinal lymph node following endobronchial ultrasound-guided transbronchial needle aspiration, a tweet was posted to invite pathologists to participate in a validation study. Twitter's direct messaging feature was used to create a group to facilitate communication among participating pathologists. Contributing pathologists reviewed consecutive cases of mediastinal lymph node resection following endobronchial ultrasound-guided transbronchial needle aspiration and examined them specifically for biopsy site changes. Data spreadsheets containing deidentified data and digital photomicrographs of suspected biopsy site changes were submitted via an online file hosting service for central review by 5 pathologists from different institutions. RESULTS.­: A total of 24 pathologists from 14 institutions in 5 countries participated in the study within 143 days of study conception, and a total of 297 cases were collected and analyzed. The time interval between study conception and acceptance of the manuscript for publication was 346 days. CONCLUSIONS.­: To our knowledge, this is the first time that a social media platform has been used to generate a research idea based on a tweet, recruit coinvestigators publicly, communicate with collaborating pathologists, and successfully complete a pathology study.

Pilomatrixoma, a Rare Mimicker of Male Breast Cancer.

Pilomatrixoma or calcifying epithelioma of Malherbe is a benign skin tumor arising from the hair follicle; breast occurrence is considered a rarity. Clinically presenting as a palpable abnormality and with both benign and malignant mammographic and sonographic features, it can be easily misdiagnosed as a breast neoplasm. We report a very rare case of pilomatrixoma of the male breast in a 36-year-old male presenting with a firm, superficial nodule in the upper outer quadrant. Though the sonographic trifecta of imaging features (shape- margins-orientation/oval, circumscribed mass, parallel to the skin) is consistent with a benign lesion, a histologic diagnosis was warranted based on its most suspicious feature of internal pleomorphic calcifications. Pathologic diagnosis revealed the uncommon benign entity of pilomatrixoma in the male breast. Our patient was recommended for surgical excision based on current literature recommendations for management in most reports of pilomatrixoma. One alternative recommendation presented in a single report of pilomatrixoma in the breast supported follow-up imaging based on benign imaging characteristics.

Development of a Novel Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein-2 That Inhibits Triple-Negative Breast Cancer and Angiosarcoma Growth In Vivo.

BACKGROUND: We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. METHODS: The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. RESULTS: Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. CONCLUSION: We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.

Specific N-Linked Glycosylation Patterns in Areas of Necrosis in Tumor Tissues.

Tissue necrosis is a form of cell death common in advanced and aggressive solid tumors, and is associated with areas of intratumoral chronic ischemia. The histopathology of necrotic regions appear as a scaffold of cellular membrane remnants, reflective of the hypoxia and cell degradation events associated with this cellular death pathway. Changes in the glycosylation of cell surface proteins is another common feature of cancer progression. Using a recently developed mass spectrometry imaging approach to evaluate N-linked glycan distributions in human formalin-fixed clinical cancer tissues, differences in the glycan structures of regions of tumor, stroma and necrosis were evaluated. While the structural glycan classes detected in the tumor and stromal regions are typically classified as high mannose or branched glycans, the glycans found in necrotic regions displayed limited branching, contained sialic acid modifications and lack fucose modifications. While this phenomenon was initially classified in breast cancer tissues, it has been also seen in cervical, thyroid and liver cancer samples. These changes in glycosylation within the necrotic regions could provide further mechanistic insight to necrotic changes in cancer tissue and provide new research directions for identifying prognostic markers of necrosis.

Increases in Tumor N-Glycan Polylactosamines Associated with Advanced HER2-Positive and Triple-Negative Breast Cancer Tissues.

PURPOSE: Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) are evaluated for N-linked glycan distribution in the tumor microenvironment. EXPERIMENTAL DESIGN: Tissue sections representing multiple human epidermal growth factor receptor 2 (HER2) receptor-positive and triple-negative breast cancers (TNBC) in both TMA and FFPE slide format are processed for high resolution N-glycan MALDI-IMS. An additional FFPE tissue cohort of primary and metastatic breast tumors from the same donors are also evaluated. RESULTS: The cumulative N-glycan MALDI-IMS analysis of breast cancer FFPE tissues and TMAs indicate the distribution of specific glycan structural classes to stromal, necrotic, and tumor regions. A series of high-mannose, branched and fucosylated glycans are detected predominantly within tumor regions. Additionally, a series of polylactosamine glycans are detected in advanced HER2+, TNBC, and metastatic breast cancer tissues. Comparison of tumor N-glycan species detected in paired primary and metastatic tissues indicate minimal changes between the two conditions. CONCLUSIONS AND CLINICAL RELEVANCE: The prevalence of tumor-associated polylactosamine glycans in primary and metastatic breast cancer tissues indicates new mechanistic insights into the development and progression of breast cancers. The presence of these glycans could be targeted for therapeutic strategies and further evaluation as potential prognostic biomarkers.

Advanced glycation end products are elevated in estrogen receptor-positive breast cancer patients, alter response to therapy, and can be targeted by lifestyle intervention.

PURPOSE: Lifestyle factors associated with personal behavior can alter tumor-associated biological pathways and thereby increase cancer risk, growth, and disease recurrence. Advanced glycation end products (AGEs) are reactive metabolites produced endogenously as a by-product of normal metabolism. A Western lifestyle also promotes AGE accumulation in the body which is associated with disease phenotypes through modification of the genome, protein crosslinking/dysfunction, and aberrant cell signaling. Given the links between lifestyle, AGEs, and disease, we examined the association between dietary-AGEs and breast cancer. METHODS: We evaluated AGE levels in bio-specimens from estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast cancer patients, examined their role in therapy resistance, and assessed the ability of lifestyle intervention to reduce circulating AGE levels in ER+ breast cancer survivors. RESULTS: An association between ER status and AGE levels was observed in tumor and serum samples. AGE treatment of ER+ breast cancer cells altered ERα phosphorylation and promoted resistance to tamoxifen therapy. In a proof of concept study, physical activity and dietary intervention was shown to be viable options for reducing circulating AGE levels in breast cancer survivors. CONCLUSIONS: There is a potential prognostic and therapeutic role for lifestyle derived AGEs in breast cancer. Given the potential benefits of lifestyle intervention on incidence and mortality, opportunities exist for the development of community health and nutritional programs aimed at reducing AGE exposure in order to improve breast cancer prevention and treatment outcomes.

Displaced Cartilage Within Lymph Node Parenchyma Is a Novel Biopsy Site Change in Resected Mediastinal Lymph Nodes Following EBUS-TBNA.

Biopsy site changes in mediastinal lymph nodes (LNs) attributable to prior endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) have not been studied in a systematic manner. Twenty-four contributors from 14 institutions in 5 countries collaborated via social media (Twitter) to retrospectively review consecutive cases of resected mediastinal LNs from patients with prior EBUS-TBNA. Resected LNs were reexamined by submitting pathologists for changes attributable to EBUS-TBNA. Patients who received neoadjuvant therapy were excluded. Cases with suspected biopsy site changes underwent central review by 5 pathologists. A total of 297 mediastinal LN resection specimens from 297 patients (183 male/114 female, mean age: 65 y, range: 23 to 87) were reviewed. Biopsy site changes were most common in station 7 (10 cases) followed by 11R, 4R, and 10R, and were found in 34/297 (11.4%) cases, including displacement of tiny cartilage fragments into LN parenchyma in 26, intranodal or perinodal scars in 7, and hemosiderin in 1. Cartilage fragments ranged from 0.26 to 1.03 mm in length and 0.18 to 0.62 mm in width. The mean interval between EBUS-TBNA and LN resection was 38 days (range: 10 to 112) in cases with biopsy site changes. A control group of 40 cases without prior EBUS-TBNA, including 193 mediastinal LN stations, showed no evidence of biopsy site changes. Biopsy site changes are identified in a subset of resected mediastinal LNs previously sampled by EBUS-TBNA. The location of the abnormalities, temporal association with prior EBUS-TBNA, and the absence of such findings in cases without prior EBUS-TBNA support the contention that they are caused by EBUS-TBNA.