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Cancer cell plasticity enables cell survival in harsh physiological environments and fate transitions such as the epithelial-to-mesenchymal transition (EMT) that underlies invasion and metastasis. Using genome-wide transcriptomic and translatomic studies, an alternate mechanism of cap-dependent mRNA translation by the DAP5/eIF3d complex is shown to be essential for metastasis, EMT, and tumor directed angiogenesis. DAP5/eIF3d carries out selective translation of mRNAs encoding EMT transcription factors and regulators, cell migration integrins, metalloproteinases, and cell survival and angiogenesis factors. DAP5 is overexpressed in metastatic human breast cancers associated with poor metastasis-free survival. In human and murine breast cancer animal models, DAP5 is not required for primary tumor growth but is essential for EMT, cell migration, invasion, metastasis, angiogenesis, and resistance to anoikis. Thus, cancer cell mRNA translation involves two cap-dependent mRNA translation mechanisms, eIF4E/mTORC1 and DAP5/eIF3d. These findings highlight a surprising level of plasticity in mRNA translation during cancer progression and metastasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The acquisition of novel detrimental cellular properties following exposure to cytotoxic drugs leads to aggressive and metastatic tumors that often translates into an incurable disease. While the bulk of the primary tumor is eliminated upon exposure to chemotherapeutic treatment, residual cancer cells and non-transformed cells within the host can engage a stable cell cycle exit program named senescence. Senescent cells secrete a distinct set of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). Upon exposure to the SASP, cancer cells undergo cellular plasticity resulting in increased proliferation, migration and epithelial-to-mesenchymal transition. The molecular mechanisms by which the SASP regulates these pro-tumorigenic features are poorly understood. Here, we report that breast cancer cells exposed to the SASP strongly upregulate Lipocalin-2 (LCN2). Furthermore, we demonstrate that LCN2 is critical for SASP-induced increased migration in breast cancer cells, and its inactivation potentiates the response to chemotherapeutic treatment in mouse models of breast cancer. Finally, we show that neoadjuvant chemotherapy treatment leads to LCN2 upregulation in residual human breast tumors, and correlates with worse overall survival. These findings provide the foundation for targeting LCN2 as an adjuvant therapeutic approach to prevent the emergence of aggressive tumors following chemotherapy., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: There is evidence that supports the association of dense tumor infiltrating lymphocyte (TILs) with an increased risk of ipsilateral recurrence in ductal carcinoma in situ (DCIS). However, the association of cellular composition of DCIS immune microenvironment with the histopathologic parameters and outcome is not well understood., Methods: We queried our institutional database for patients with pure DCIS diagnosed between 2010 and 2019. Immunohistochemical studies for CD8, CD4, CD68, CD163, and FOXP3 were performed and evaluated in the DCIS microenvironment using tissue microarrays. Statistical methods included Fisher's exact test for categorical variables and the two-sample t-test or the Wilcoxon Rank-Sum test for continuous variables., Results: The analytic sample included 67 patients. Median age was 62 years (range = 53 to 66) and median follow up was 6.7 years (range = 5.3 to 7.8). Thirteen patients had ipsilateral recurrence. Of all the clinicopathologic variables, only the DCIS size and TIL density were significantly associated with recurrence (p = 0.023 and 0.006, respectively). After adjusting for age and TIL density, only high CD68 (>50) and high CD68/CD163 ratio (>0.46) correlated with ipsilateral recurrence (p = 0.026 and 0.013, respectively) and shorter time to recurrence [hazard ratio 4.87 (95% CI: 1.24-19, p = 0.023) and 10.32 (95% CI: 1.34-80, p = 0.025), respectively]., Conclusions: In addition to DCIS size and TIL density, high CD68 + tumor-associated macrophages predict ipsilateral recurrence in DCIS. High CD68 + macrophage density and CD68/CD163 ratio also predict a shorter time to recurrence., (Copyright © 2022. Published by Elsevier Ltd.)

The management of intraductal papilloma (IDP) diagnosed on core needle biopsy (CNB) is controversial due to the variable upgrade rates to breast carcinoma (BC) on subsequent surgical excision reported in the literature. The purpose of our study was to investigate the upgrade rate of IDP diagnosed on CNB to BC in subsequent surgical excision and the impact of clinical, pathologic, and radiologic variables. This is a retrospective cohort of all women who had a diagnosis of IDP on a CNB between 2005 and 2018 in a tertiary academic center with subsequent surgical excision. Upgrade was defined as ductal carcinoma in situ (DCIS) and invasive carcinoma on surgical excision. Statistical analyses included Pearson's chi-square, Wilcoxon rank-sum, and logistic regression. A total of 216 women with IDP in a CNB were included. Nineteen patients (8.8%) upgraded to BC in the overall cohort, including 14 DCIS and 5 invasive carcinomas. An upgrade rate of 27% was found in atypical IDP (14 of 51 cases), while only 3% of pure IDP upgraded to BC (5 of 165 cases). Older age (>53 years) at the time of biopsy (odds ratio [OR] = 1.05, 95% confidence interval [CI] = 1.01-1.09, p = 0.027) and concomitant atypical ductal hyperplasia (ADH) (OR = 9.69, 95% CI = 3.37-27.81, p < 0.0001) were significantly associated with upgrade. Our results support surgical excision of IDP on CNB when associated with ADH or diagnosed in women aged older than 53 years. The low surgical upgrade rate of 3% for pure IDP on CNB in younger women should be part of the management discussion., (Copyright © 2020 Elsevier Inc. All rights reserved.)

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies., Competing Interests: Disclosures: C.J.R. Foster reported grants from the National Institutes of Health during the conduct of the study. K-K. Wong reported "other" from G1 Therapeutics, Zentalis Therapeutics, and Epiphanes Therapeutics outside the submitted work, and has consulting/sponsored research agreements with the following: AstraZeneca, Janssen, Pfizer, Novartis, Merck, Ono, and Array (consulting and sponsored research); MedImmune, Mirati (which developed MRTX 1257), Takeda, TargImmune, and BMS (sponsored research only). B.G. Neel reported "other" from Navire Pharma, Northern Biologics, Ltd, Arvinas, Inc, Regeneron, Amgen, Inc, Mirati Therapeutics, Gilead Therapeutics, and Moderna outside the submitted work. In addition, B.G. Neel has a patent to PCT 63031457 pending. No other disclosures were reported., (© 2020 Fedele et al.)

In diagnostic breast pathology, there is no reliable applicable immunostain to help discern atypical and in situ apocrine lesions from benign apocrine tissue. At present, the diagnosis of non-invasive apocrine lesions remains challenging with current diagnoses rendered based on discrete morphologic characteristics on conventional hematoxylin and eosin staining. Interobserver variability is significant even among subspecialists partly due to lack of adjuvant diagnostic immunohistochemical stains. Herein, we set to elucidate the potential utility of EZH2 and Ki-67 immunostains as tangible tools in non-invasive apocrine proliferations. A cohort of apocrine breast lesions [Benign apocrine hyperplasia (BAH), n = 10; Atypical apocrine hyperplasia (AAH), n = 16; Apocrine ductal carcinoma in situ (ADCIS), n = 12] were subjected to EZH2 immunostaining and analyzed via H-scoring of nuclear expression. Mean H-scores for EZH2 progressively increased from BAH (23.5), to AAH (47.4) and ADCIS (196.4), and showed a significant difference utilizing the Kruskal-Wallis test (p < 0.0001). Further interrogation of Ki-67 demonstrated incremental expression from BAH to AAH and ADCIS at 1.6 %, 4.7 % and 24.7 %, respectively (p < 0.0001, Kruskal-Wallis test), suggesting an association with increased proliferation. Our results demonstrate that a combination of EZH2 and Ki-67 immunostaining may be employed in differentiating among challenging apocrine breast lesions and suggest a putative diagnostic utility for EZH2 and Ki-67 in non-invasive apocrine breast lesions., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Context.—: Bilateral mastectomy for chest masculinization is one of the gender-affirming procedures for transmasculine individuals., Objective.—: To optimize gross handling protocols and assess histopathologic findings in transmasculine breast tissue specimens., Design.—: We identified all gender-affirming mastectomies from 2015 to 2018. We sequentially identified reduction mammoplasty (RM) cases for macromastia from the same period as control. Significant findings were defined as atypical ductal or lobular hyperplasia (ADH, ALH), ductal or lobular carcinoma in situ (DCIS, LCIS), or invasive carcinoma., Results.—: Significant findings were present in 6 of 211 gender-affirming mastectomies (2.8%) as follows: ADH (n = 5) and LCIS together with ALH (n = 1). By comparison, 19 of 273 RM specimens (7%) yielded significant findings as follows: ALH (n = 11), ADH (n = 4), LCIS (n = 2), DCIS (n = 1), and invasive lobular carcinoma (n = 1). In the gender-affirming group, 142 transmen underwent androgen therapy before surgery, of whom 2 had significant pathologic findings. Thirty and 41 individuals had a family history of breast cancer in the gender-affirming and RM group, of whom 1 and 3 individuals had significant pathologic findings, respectively., Conclusions.—: Our study demonstrates that we handle transmasculine mastectomy specimens by examining 2.8 times more slides on average than for RMs, with a 2.5 times lower rate of significant pathologic findings. Prior family history of breast cancer or the use of androgen therapy before surgery in gender-affirming individuals did not increase the risk of identifying significant breast lesions. We recommend submitting 4 tissue blocks per mastectomy for individuals undergoing gender-affirming breast surgery., (© 2020 College of American Pathologists.)

Amyloidosis is characterized by extracellular deposition of insoluble protein fibrils in a beta-pleated sheet configuration. Breast amyloidosis is a rare entity which has previously been reported to present with localized involvement, or as a late manifestation of systemic amyloidosis. However, descriptions of the clinicopathologic features of localized breast amyloidosis remain limited. A retrospective search for breast amyloidosis diagnosed at our institution yielded 10 cases of breast amyloidosis. All patients were female, with a mean age of 69. Median follow-up for survival or progression was 13 months. Indications for breast or axilla biopsy included mammographic calcifications, mass, and axillary lymphadenopathy. Amyloid showed positive staining with Congo red in all cases, and amyloid typing revealed light chain lambda in 3 cases, amyloid transthyretin in 2 cases, light chain kappa in 1 case, and iatrogenic insulin-derived amyloidosis in 1 case. Amyloid occurred within axillary lymph nodes and alongside both benign and neoplastic breast tissue, including atypical ductal hyperplasia, lobular carcinoma in situ and ductal carcinoma in situ. Most cases were associated with predisposing clinical conditions, including autoimmune disease in 4 cases, B cell lymphomas in 2 cases, and diabetes mellitus treated with insulin in 1 case. In contrast to previously published case series, no patient had clinical evidence of systemic amyloidosis. Amyloidosis of the breast should be considered in the differential diagnosis of all mammographic calcifications and masses of the breast or axilla. When recognized correctly on biopsy, the diagnosis of amyloidosis can not only prevent further unnecessary surgical interventions due to radiology-pathology discordance, but initiate the necessary amyloidosis work-up. Although rare, an awareness of the clinicopathologic characteristics of this easily overlooked entity is of great importance for every practicing pathologist reviewing breast biopsies., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: Growing evidence suggests that the tumor immune microenvironment influences breast cancer development and prognosis. Density of tumor-infiltrating lymphocytes (TILs) within invasive breast cancer is correlated with response to therapy, especially in triple-negative disease. The clinical relevance and outcomes of TILs within ductal carcinoma in situ (DCIS) are less understood., Methods: Our institutional database of 668 patients with pure DCIS from 2010 to 2018 was queried. TILs were evaluated by International TILs Working Group guidelines. Percentage of TILs was assessed from the densest focus (hotspot) in one high-power field of stroma touching the basement membrane. Statistical methods included cluster analyses (to define sparse versus dense TILs), logistic, and Cox regression models., Results: Sixty-nine patients with DCIS and TILs were evaluated, of whom 54 (78%) were treated by breast-conserving surgery. Thirteen (19%) patients had ipsilateral recurrence. Each recurrence (n = 13) was matched to four controls (n = 56) based on date of surgery. Median follow-up was 6.7 years. TILs were defined as sparse (< 45%) or dense (≥ 45%). Dense TILs were associated with younger age (p = 0.045), larger tumor size (p < 0.001), high nuclear grade (p = 0.010), comedo histology (p = 0.033), necrosis (p = 0.027), estrogen receptor (ER) negativity (p = 0.037), and ipsilateral recurrence (p = 0.001). Nine patients with dense TILs had mean time to recurrence of 73.5 months compared with four patients with sparse TILs with mean time to recurrence of 97.9 months (p = 0.003)., Conclusions: Dense TILs were significantly associated with age, tumor size, nuclear grade, comedo histology, necrosis, and ER status and was a significant predictor of recurrence in patients with pure DCIS.

Adaptive resistance to MEK inhibitors (MEKi) typically occurs via induction of genes for different receptor tyrosine kinases (RTK) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 ( PTPN11 ) is required for RAS/ERK pathway activation by most RTKs and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEKi inhibited the proliferation of multiple cancer cell lines in vitro PTPN11 knockdown/MEKi treatment had similar effects, whereas expressing SHP099 binding-defective PTPN11 mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of KRAS -mutant pancreas, lung, and ovarian cancers and in wild-type RAS-expressing triple-negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEKi, and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEKi combinations could have therapeutic utility in multiple malignancies. Significance: MEK inhibitors show limited efficacy as single agents, in part because of the rapid development of adaptive resistance. We find that SHP2/MEK inhibitor combinations prevent adaptive resistance in multiple cancer models expressing mutant and wild-type KRAS. Cancer Discov; 8(10); 1237-49. ©2018 AACR. See related commentary by Torres-Ayuso and Brognard, p. 1210 This article is highlighted in the In This Issue feature, p. 1195 ., (©2018 American Association for Cancer Research.)

Background: In this study, the pattern of distribution of the nuclei immunoreactive with Ki67 was examined in DIN1c (DCIS, grade 1/low grade), DIN2 (DCIS, grade 2/intermediate grade), and DIN3 (DCIS, grade 3/high grade). The lesions were evaluated to determine if distinctive patterns could be identified in correlation with lesion grade., Methods: Fifty seven (n=57) consecutive DIN cases were investigated. Of these, 15 qualified as DIN1c, 28 as DIN2 and 14 as DIN3. The patterns of distribution were recorded for each case as either basal/peripheral or haphazard within the epithelial proliferation., Results: There was a statistically significant difference between the DIN1c, DIN2 and DIN3 in terms of basal/peripheral versus haphazard distribution of Ki67 immunostaining (Chi-square test, P<0.0001). Basal/peripheral staining pattern was dominant among the DIN1c cases, while haphazard staining pattern was the dominant distribution among the DIN3 cases. One half of the DIN2 cases showed basal/peripheral staining pattern, while the other half showed a haphazard staining pattern., Conclusion: High grade DIN lesions show haphazard Ki67 staining while low grade DIN lesions show basal/peripheral Ki67 staining in the proliferating epithelial cells. This feature could be practical in separating DIN lesions into low grade (basal/peripheral-Ki67) and high grade (haphazard-Ki67) eliminating the grade 2/intermediate category., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Pathologists should be aware of the existence of a rare CK7-negative variant of breast carcinoma in general, and of Paget's disease in particular. Cytokeratin 7-negative Paget's disease and CK7-negative ductal intraepithelial neoplasia (ductal carcinoma in situ) present a major diagnostic challenge for pathologists since there is limited awareness of their existence. When there is classic Paget's morphology on H&E sections, GATA3 positivity should resolve any doubts about the diagnosis in the setting of a CK7-negative neoplastic cell population., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A 52-year-old otherwise healthy woman presented with a solitary firm mass in the right breast. Histopathological evaluation of the 1.5-cm mass showed a mammary myofibroblastoma of the conventional spindle-cell type. High-power examination of hematoxylin-eosin-stained sections showed round, eosinophilic, intracytoplasmic, as well as extracellular, hyaline globules. These 5- to 20-µm globules appeared gray with a pinkish rim on Masson's trichrome stain. Immunohistochemically, the hyaline globules were strongly reactive with smooth muscle myosin heavy chain, desmin, and caldesmon. Histologically similar inclusion bodies have been reported in phylloides tumors--including those with myoid differentiation. To our knowledge, this is the first description of hyaline globules, a peculiar histological curiosity with no known clinical significance, in mammary myofibroblastoma., (© The Author(s) 2014.)

Eccrine spiradenomas are uncommon, benign lesions, which are thought to originate from the eccrine sweat glands. They are common in young adults and are without a sex predilection. Here we report a case of eccrine spiradenoma of the breast skin in a 39-year-old woman who presented with a breast nodule for 10 years. It is crucial to take eccrine spiradenoma into consideration in superficial, well-circumscribed, breast skin/subcutaneous lesions. It is useful to recognize the two-cell populations constituting this tumor: small, dark, basaloid cells with hyperchromatic nuclei, which are immunoreactive for P63 and calponin, and larger cells with a pale nucleus, often near the center of the cluster (inner cells), which are immunoreactive for CK7 and CD117 (C-kit).

Size (the "T" in the TNM System) of invasive breast carcinoma is a proven independent prognostic factor; however, its accurate determination can be challenging. The purpose of this review is to discuss the complexities inherent in determining "T"-including those encountered in the clinical measurement ("cT", ie, physical and radiologic assessment) as well as pathologic determination (pT) of invasive breast carcinomas. Pathologic estimation of tumor size, macroscopic, as well as microscopic, can be problematic due to the complexity of multiple situations, seeming confusion regarding staging guidelines, and interobserver variation in interpretation. Additional problematic scenarios in determination of "T" include those incurred in excisions performed after the performance of needle core biopsies, and in cases wherein there are multiple foci of invasive carcinoma, as well as in carcinomas status post-neoadjuvant chemotherapy. It can also be difficult to determine "T" in certain types of invasive carcinoma, particularly those of the lobular type. In this communication, some of the complexities and challenges in determing "T" are discussed, and modest suggestions are offered to assist in optimizing such assessments.

Although Paget disease of the nipple (PDN) is a well-established clinical and pathological neoplastic process, invasive PDN (IPDN) is a relatively newly described disease. The latter entity is characterized by invasive carcinoma that is localized to the nipple and is associated with PDN as well as with either intraductal and/or invasive carcinoma in the underlying breast. To our knowledge, only 17 cases of IPDN, all node negative, have been reported. Here, we report the case of a 68-year-old woman with invasive Paget disease of the left nipple. The patient had a history of intraductal carcinoma, treated by lumpectomy alone. She presented 6 years later with "eczematous" lesion of the ipsilateral nipple, a punch biopsy of which showed a superficially IPDN as well as conventional PDN. The subsequently performed wide excision of the nipple, areola, and underlying breast tissue showed the invasive carcinoma to span 0.6 cm. Then, 3 months later, the patient presented with ipsilateral palpable axillary lymphadenopathy. Axillary dissection revealed metastatic carcinoma in 7 of 19 lymph nodes. This case of IPDN not only represents the deepest extent of invasion reported thus far but also the only one known to be node positive., (© The Author(s) 2014.)

Aims: There are scant data on the correlation of maximum tumor dimension (MTD) in needle core biopsy (NCB) and in subsequent excisional biopsy (EXB) with various pre-NCB imaging studies (VIS)-especially in the context of screen-detected invasive carcinoma (SIC)., Methods and Results: Retrospectively studied were consecutive (2012-2013) non-palpable, SIC diagnosed on NCB with subsequent EXB. Data on MTD on VIS (either mammogram, or ultrasound, or MRI), NCB and EXC were analyzed. Mean MTD on VIS was 12.5mm (range: 0-45 mm). Mean MTD on NCB was 6.7 mm (range: 1-15 mm). Mean residual MTD on EXB was 12.9 mm (range: 0-40 mm). Mean number of NCB performed per SIC was 5 (range: 1-13). Overall, 81% of all NCB were involved by SIC. The difference between MTD at EXC and VIS was statistically not significant (p>0.05). Spearman correlation coefficient for MTD on VIS and EXC was r=0.8718 (p<0.0001) showing a significant correlation. The mean tissue volume procured on NCB-calculated by using Aperio whole slide scanning and NIH Image J image analysis was 95.5mm(3) (range: 4.3-887.5mm(3), median: 23 mm(3)). A Bland-Altman plot showed that MTD of ≥ 7 mm on EXB is a useful cut-off point predictive of (any) increase in MTD at EXB. Six of the 13 patients with MTD< 7mm on EXB showed a decrease in size; while no patient with MTD on EXB that was ≥ 7 mm showed any decrease in size. (Fisher's exact test, P=0.001, two-tailed). Overall 88% (44 out of 50 patients) of SIC showed no decrease in MTD on EXB, with an increase by ≥ 4 mm in size (sufficient to upstage "T") in MTD of ≥ 7mm on EXB in 75.6% (28 of 37 patients with MTD of ≥ 7 mm on EXB). 20.8% of SIC (5 of 24 patients) that were < 7 mm on NCB (with a mean combined Nottingham grade score of 5 {r: 4-6} showed decrease in MTD at EXB., Conclusions: In this pilot study of SIC, (i) MTD on VIS was predictive of MTD on EXB, (ii) MTD of ≥ 7 mm on NCB was predictive of an increased MTD on EXB in most cases, with potential for "upstaging" tumors, and (iii) MTD of < 7 mm on NCB was predictive of decreased MTD on EXB in 20.8% of (mostly grade I) SIC. Procured tissue volume on NCB contributed to decrease in MTD on EXB in small, low-grade carcinomas., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Due to the proximity of the skin, subcutis, and axilla to the breast, the possibility of a "breast mass" actually representing a dermatologic lesion should be considered, particularly if the proliferation does not look characteristically "mammary" in appearance. Even more underappreciated is the scenario of a dermatologic proliferation morphologically masquerading as a breast tumor. The pathologist can fall prey to this pitfall if he/she is led to believe that the location of the tumor is the breast proper. The aim of this review is to provide an overview of dermatologic mimickers of breast lesions and helpful ways to discern between them when possible.

Unlabelled: Phyllodes tumors (PT) are rare fibroepithelial breast tumors representing less than 1 % of all breast malignancies. These tumors are unpredictable and fast growing with a high local recurrence rate, making this disease challenging to treat. Previous literature focused on surgical resection, and breast reconstruction following a mastectomy in patients with PT is rarely addressed. We report a case of a recurrent malignant PT treated with a nipple-sparing mastectomy followed by immediate single-stage silicone implant breast reconstruction. While PT is a rare breast malignancy that presents challenges with both surgical resection and reconstruction, we demonstrate that nipple-sparing mastectomy with immediate implant breast reconstruction with AlloMax is curative and can offer an appealing cosmetic option., Level of Evidence V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Aims: Quantitative image analysis of histopathology slides is becoming an important technology in diagnostic pathology. To this end, it is essential to combine a robust image analysis software with the most commonly used immunohistochemical staining methods. In this investigation, we describe a practical application of NIH ImageJ software for quantitative vascular image analysis for diaminobenzene chromogen-based CD34 immunostain in breast cancer. CD34 immunostain is in a unique position to identify lymphangiogenesis and angiogenesis simultaneously in a given tumor tissue. This investigation aims at establishing a practical quantitative vascular image analysis solution for diagnostic pathologists by using ImageJ, and CD34 immunostain., Methods and Results: Tissue microarray slides containing breast cancer tissue were immunostained for CD34 for simultaneous identification of lymphatic endothelial cells (LEC) and blood vessel endothelial cells (BEC). Digital images were analyzed using NIH ImageJ software. A CD34 score was quantified for each tissue core as a percentage (CD34-positive area/area of tissue core). The mean CD34 scores were 0.24%, 0.40%, 1.30%, 2.33%, 2.64%, and 3.44% for normal breast tissue, in stage IIA, IIB, IIIA, IIIB, and IIIC breast cancer tissue cores, respectively (p<0.0001). The mean CD34 scores were 0.70% and 2.21% for lymph node-negative and lymph node-positive breast cancer patients, respectively (p<0.0001)., Conclusions: ImageJ software seems to be an attractive quantitative image analysis tool for diagnostic pathology for immunohistochemistry-based applications because of its capabilities, availability, and ease of use with most image formats. Our results show the feasibility, versatility, and ease of use of ImageJ and CD34 immunohistochemistry for vascular image analysis in breast pathology. Given the prospects of novel lymphatic and vascular endothelium-targeting therapeutics in breast oncology, the practical analysis of combined LEC and BEC density described in this report could enable diagnostic pathologists to apply quantitative vascular image analysis easily in their pathology practice and translational research., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The walls of angiogenic blood vessel capillaries are composed of two principal cell types, blood vessel endothelial cells (BEC) and pericytes (PC), whereas the walls of lymphatic capillaries are composed of lymphatic endothelial cells (LEC). In this investigation we describe a practical application of NIH ImageJ software for quantitative image analysis for pericytes and endothelial cells in prostate cancer. We used a tissue microarray that contained 49 tissue cores (normal prostate tissue or prostatic carcinomas with Gleason scores of 6 through 10). These prostate cancer samples represented AJCC prognostic stages II, III, and IV. Slides were immunostained with anti-PDGFR-β antibody for identification of PC, and quantified as microvascular pericyte density (MVPD); they were also immunostained with anti-CD34 antibody for identification of LEC and BEC simultaneously, and quantified as microvascular endothelial density (MVED). CD31 and D2-40 immunostains were used to quantify BEC and lymphatic endothelial cells, respectively. Our results showed higher MVPD and MVED in prostate cancers with higher Gleason scores and higher stages, suggesting the prognostic utility of vascular image analysis in prostate pathology. This investigation demonstrates the feasibility, versatility, and ease of use of ImageJ software and pericyte-specific and endothelial-specific immunohistochemistry for quantitative image analysis in prostate pathology., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Sprouting of angiogenic perivascular cells is thought to be highly dependent upon autocrine and paracrine growth factor stimulation. Accordingly, we report that corneal angiogenesis induced by ectopic FGF implantation is strongly impaired in NG2/CSPG4 proteoglycan (PG) null mice known to harbour a putative deficit in pericyte proliferation/mobilization. Conversely, no significant differences were seen between wild type and knockout corneas when VEGF was used as an angiocrine factor. Perturbed responsiveness of NG2-deficient pericytes to paracrine and autocrine stimulation by several FGFs could be confirmed in cells isolated from NG2 null mice, while proliferation induced by other growth factors was equivalent in wild type and knockout cells. Identical results were obtained after siRNA-mediated knock-down of NG2 in human smooth muscle-like cell lines, as also demonstrated by the decreased levels of FGF receptor phosphorylation detected in these NG2 deprived cells. Binding assays with recombinant proteins and molecular interactions examined on live cells asserted that FGF-2 bound to NG2 in a glycosaminoglycan-independent, core protein-mediated manner and that the PG was alone capable of retaining FGF-2 on the cell membrane for subsequent receptor presentation. The use of dominant-negative mutant cells, engineered by combined transduction of NG2 deletion constructs and siRNA knock-down of the endogenous PG, allowed us to establish that the FGF co-receptor activity of NG2 is entirely mediated by its extracellular portion. In fact, forced overexpression of the NG2 ectodomain in human smooth muscle-like cells increased their FGF-2-induced mitosis and compensated for low levels of FGF receptor surface expression, in a manner equivalent to that produced by overexpression of the full-length NG2. Upon FGF binding, the cytoplasmic domain of NG2 is phosphorylated, but there is no evidence that this event elicits signal transductions that could bypass the FGFR-mediated ones. Pull-down experiments, protein-protein binding assays and flow cytometry FRET coherently revealed an elective ligand-independent association of NG2 with FGFR1 and FGFR3. The NG2 cooperation with these receptors was also corroborated functionally by the outcome of FGF-2 treatments of cells engineered to express diverse NG2/FGFR combinations. Comprehensively, the findings suggest that perivascular NG2 may serve as a dual modulator of the availability/accessibility of FGF at the cell membrane, as well as the resulting FGFR transducing activity.

In this report we describe a practical procedure for measuring interstitial fluid pressure (IFP) using fiberoptic pressure transducers based on optical interferometry. Eight mice were used for subcutaneous IFP measurements and four mice for intramuscular IFP measurements with a FOBPS-18 fiberoptic pressure transducer. We used four mice for subcutaneous IFP measurements with a SAMBA-420 MR fiberoptic pressure transducer. One measurement was made for each mouse simultaneously by using a fiberoptic system and an established approach, either transducer-tipped catheter or wick-in-needle technique. The mean IFP values obtained in subcutaneous tissues were -3.00 mm Hg (SEM-/+0.462, n=8), -3.25 mm Hg (SEM-/+0.478, n=4), -3.34 mm Hg (SEM-/+0.312, n=6), and -2.85 (SEM-/+0.57, n=6) for the FOBPS fiberoptic transducer, the SAMBA fiberoptic transducer, the transducer-tipped catheter, and the wick-in-needle technique, respectively. There was no difference between these techniques to measure IFP (Friedman test, p=0.7997). The subcutaneous IFP measurements showed strong linear correlation between fiberoptic transducer and transducer-tipped catheter (R(2)=0.9950) and fiberoptic transducer and wick-in-needle technique (R(2)=0.9966). Fiberoptic pressure transducers measure the interstitial fluid pressure accurately, comparable to conventional techniques. The simplified IFP measurement procedures described in this report will allow investigators to easily measure IFP, and elucidate the unit pressure change per unit volume change (dP/dV) in normal or cancer tissues in the presence of strong electromagnetic fields encountered in MRI.

Purpose: The aim of this study is to investigate markers of inflammation and oxidative stress in an early model of diabetic retinopathy, correlate retinal and plasma results and evaluate the influence of treatment by N-acetylcysteine (NAC), a free radical scavenger., Methods: Four groups were studied: control (C), streptozotocin (STZ)-induced diabetic rats (D), STZ rats following 8 weeks of NAC (DT), and control rats following 8 weeks of NAC (CT). Plasma levels of free 15-F2t-isoprostane (15-F-2t-IsoP), superoxide dismutase (SOD) and tumour necrosis factor-alpha (TNF-alpha) were obtained. Primary antibodies against macrophages (ED-1), microglia (Ox-42), pericytes (NG-2), endothelial and perivascular cells (IB-4), haem oxygenase 1 (HO-1) and vascular endothelial growth factor (VEGF) were used., Results: Expression of NG-2 was robust in C, CT, DT, and mild in D. The intensity of IB-4 was higher in D and DT compared with the C and CT. Ox-42 and ED-1 expression was higher in the D than in the DT, C or CT. Expression of VEGF and HO-1 was non-specific across the four groups. Plasma levels of 15-F-2t-IsoP and TNF-alpha were higher in the D as compared with the C, CT and DT. SOD levels were lower in the D when compared with the C, CT and D., Conclusions: Macrophage/microglia activation, pericyte loss and endothelial/perivascular cell changes occur early in the pathogenesis of DR. These changes are associated with an increase in plasma markers of oxidative stress and inflammation and are minimized by treatment with NAC. The results suggest that therapies that reduce free radicals will help minimize the early events in diabetic retinopathy in the STZ model.

Research on vascular adaptation to microgravity in the central nervous system requires a simple, noninvasive, direct imaging technique that can be performed with compact equipment. In this report we describe a practical, nonmydriatic, retinal self-imaging technique using a Kowa Genesis-D hand-held digital camera and a Black and Decker laser level. This simple technique will be useful to clinical physiologists conducting microgravity research, as well as for the studies of high-altitude medicine and aviation physiology.

Context: The transgenic human islet amyloid polypeptide (HIP) rat model of type 2 diabetes mellitus (T2DM) parallels the functional and structural changes in human islets with T2DM., Objective: The transmission electron microscope (TEM) was utilized to observe the ultrastructural changes in islet microcirculation., Methods: Pancreatic tissue from male Sprague Dawley rats (2, 4, 8, 14 months) were used as controls (SDC) and compared to the 2-, 4-, 8- and 14-month-old HIP rat models., Results: The 2-month-old HIP model demonstrated no islet or microcirculation remodeling changes when compared to the SDC models. The 4-month-old HIP model demonstrated significant pericapillary amyloid deposition and diminution of pericyte foot processes as compared to the SDC models. The 8-month-old model demonstrated extensive islet amyloid deposition associated with pericyte and beta-cell apoptosis when compared with SDC. The 14-month-old HIP model demonstrated a marked reduction of beta-cells and intra-islet capillaries with near complete replacement of islets by amyloidoses. Increased cellularity in the region of the islet exocrine interface was noted in the 4- to 14-month-old HIP models as compared to SDC. In contrast to intra-islet capillary rarefaction there was noticeable angiogenesis in the islet exocrine interface. Pericytes seemed to be closely associated with collagenosis, intra-islet adipogenesis and angiogenesis in the islet exocrine interface., Conclusion: The above novel findings regarding the microcirculation and pericytes could assist researchers and clinicians in a better morphological understanding of T2DM and lead to new strategies for prevention and treatment of T2DM.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Background: The walls of capillaries in prostate cancer are composed of endothelial cells, and pericytes. NG2 is a transmembrane proteoglycan on nascent pericytes with a functional role in neovascularization., Methods: The anti-angiogenic effect of hydron pellets containing NG2 neutralizing antibody was quantified in intracorneal PC-3 and LNCaP xenografts. TRAMP and TRAMP-C1 tumors grafted in NG2 knockout mice represented intrinsic pericyte targeting. TRAMP and TRAMP-C1 grafts were analyzed with confocal microscope for microvascular density (MVD) and lymphatic vascular density (LVD)., Results: NG2 neutralizing antibody decreased corneal neovascularization in PC3 (P<0.0001), and LNCaP (P=0.0079) xenografts. Mean MVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 71% (P=0.0006) and 63% (P=0.0011) lower than wild type controls, respectively. Mean LVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 73% (P=0.0003) and 84% (P<0.0001) lower than wild type controls, respectively., Conclusions: Targeting of pericyte-NG2 decreases neovascularization and lymphangiogenesis in prostate cancer significantly., (Copyright (c) 2005 Wiley-Liss, Inc.)

In this investigation, we explored whether knockout of nerve/glial antigen 2 (NG2), a pericyte component, inhibited neovascularization and growth of uveal melanoma xenografts. For this, we used multichannel laser scanning confocal microscopy and quantitative image analysis. Orthotopic human uveal melanoma (OCM-1A) xenografts were induced in NG2 knockout and wild-type mice, which were immunosuppressed with cyclosporin A. Inhibition of pericytes through NG2 proteoglycan decreased neovascularization and tumor end volume, rendering pericytes and NG2 proteoglycan potential cellular and molecular therapeutic targets in uveal melanoma., (Copyright (c) 2006 S. Karger AG, Basel.)

Purpose: Bone-marrow (BM)-derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization., Methods: A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)-positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software., Results: BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM., Conclusions: Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.

Bone marrow (BM)-derived cells are thought to participate in the growth of blood vessels during postnatal vascular regeneration and tumor growth, a process previously attributed to stem and precursor cells differentiating to endothelial cells. We used multichannel laser scanning confocal microscopy of whole-mounted tissues to study angiogenesis in chimeric mice created by reconstituting C57BL mice with genetically marked syngeneic BM. We show that BM-derived endothelial cells do not significantly contribute to tumor- or cytokine-induced neoangiogenesis. Instead, BM-derived periendothelial vascular mural cells were persistently detected at sites of tumor- or vascular endothelial growth factor-induced angiogenesis. Subpopulations of these cells expressed the pericyte-specific NG2 proteoglycan, or the hematopoietic markers CD11b and CD45, but did not detectably express the smooth muscle markers smooth muscle alpha-actin or desmin. Thus, the major contribution of the BM to angiogenic processes is not endothelial, but may come from progenitors for periendothelial vascular mural and hematopoietic effector cells.