Congenital infection of SARS-CoV-2 appears to be exceptionally rare despite many cases of COVID-19 during pregnancy. Robust proof of placental infection requires demonstration of viral localization within placental tissue. Only two of the few cases of possible vertical transmission have demonstrated placental infection. None have shown placental expression of the ACE2 or TMPRSS2 protein, both required for viral infection. We examined 19 COVID-19 exposed placentas for histopathologic findings, and for expression of ACE2, and TMPRSS2 by immunohistochemistry. Direct placental SARS-CoV-2 expression was studied by two methods-nucleocapsid protein expression by immunohistochemistry, and RNA expression by in situ hybridization. ACE2 membranous expression in the syncytiotrophoblast (ST) of the chorionic villi is predominantly in a polarized pattern with expression highest on the stromal side of the ST. In addition, cytotrophoblast and extravillous trophoblast express ACE2. No ACE2 expression was detected in villous stroma, Hofbauer cells, or endothelial cells. TMPRSS2 expression was only present weakly in the villous endothelium and rarely in the ST. In 2 of 19 cases, SARS-CoV-2 RNA was present in the placenta focally in the ST and cytotrophoblast. There was no characteristic histopathology present in our cases including the two placental infections. We found that the placenta is capable of being infected but that this event is rare. We propose one explanation could be the polarized expression of ACE2 away from the maternal blood and pronounced paucity of TMPRSS2 expression in trophoblast.

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.nonlinear microscopy | breast cancer | pathology | imaging B reast cancer is the most common cancer and cause of cancerrelated death in women worldwide (1). In the United States, one in eight women will develop breast cancer in their lifetimes with an estimated 226,870 new cases and 39,510 deaths in 2012 (2). Breast-conserving therapy (BCT), lumpectomy followed by adjuvant radiation and/or chemotherapy and hormonal therapy, is a standard of care for early breast cancer and achieves survival and local recurrence rates comparable to mastectomy while providing superior cosmetic outcomes and reduced morbidity (3-5). Local recurrence rates are correlated with surgical margin status, which is evaluated using intraoperative examination and postoperative histopathology. However, there is no consensus on surgical margin widths, and differing conventions for negative and close margins have shown inconsistent correlations with therapeutic outcomes (6-9). Despite these inconsistencies, margins initially positive for invasive cancer or ductal carcinoma in situ (DCIS) almost always require surgical reexcision (92.6% when positive for DCIS and 83.5% when positive for invasive cancer) (9), and up to 40% of all women undergoing BCT require repeat surgeries from positive or close margins (10-12). In addition to surgical complications and incr...

The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. GATA3 in particular is necessary for mammary gland maturation, and its loss has been implicated in breast cancer development. Our goal was to validate the ability of GATA3 expression to predict survival in breast cancer patients. Protein expression of GATA3 was analyzed on a high density tissue microarray consisting of 242 cases of breast cancer. We associated GATA3 expression with patient outcomes and clinicopathological variables. Expression of GATA3 was significantly increased in breast cancer, in situ lesions, and hyperplastic tissue compared to normal breast tissue. GATA3 expression decreased with increasing tumor grade. Low GATA3 expression was a significant predictor of disease-related death in all patients, as well as in subgroups of estrogen receptor positive or low grade patients. Additionally, low GATA3 expression correlated with increased tumor size and estrogen and progesterone receptor negativity. GATA3 is an important predictor of disease outcome in breast cancer patients. This finding has been validated in a diverse set of populations. Thus, GATA3 expression has utility as a prognostic indicator in breast cancer.

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The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 downregulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its downregulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER þ breast cancer cell proliferation. MFG-E8 also decreased in erbB2 þ human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple-negative (ER À , PgR À , erbB2 À ) breast cancers, cell lines, and patient sera. Knockdown, chromatin immunoprecipitation, and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple-negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple-negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER þ and erbB2 þ breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple-negative breast cancers urges continued evaluation of its differential functions. Cancer Res; 71(3); 937-45. Ó2010 AACR.

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The commonly used Nottingham Grading System in breast cancer takes into consideration the presence of tubular formation, nuclear pleomorphism, and the mitotic index (MI), among which the latter has been shown to be the most powerful prognostic factor. In practice, histologic grading is highly subjective, with only moderate interobserver reproducibility. Phosphorylation of histone H3 has been demonstrated to be a specific event in the mitotic phase, and is negligible during interphase. In this study, we evaluated the efficacy of Phosphohistone H3 (PHH3) in the breast cancer grading of 97 consecutive biopsy specimens. PHH3 antibodies clearly revealed discrete, strong nuclear immunoreactivity in mitotically active cells even under low magnification. The PHH3 MI showed a significant correlation with that derived by hematoxylin and eosin (H&E) staining as well as the Ki-67 proliferation index. Further, the pairwise κ-value of the MI was significantly increased, and the pairwise agreement was also markedly improved by PHH3 immunostaining, although a significant proportion of breast cancer cases were upgraded by use of the PHH3 MI. Our data showed that PHH3 provided a more sensitive and accurate MI with less interobserver variability when compared with conventional H&E staining, thus emphasizing its potentially increased value in practice. Reconsideration of breast cancer grading with integration of PHH3 should be considered if it continues to demonstrate superiorly to traditional H&E staining.

It is known that V-ATPases (vacuolar H+-ATPase) are involved in breast cancer growth and metastasis. Part of this action is similar to their role in osteoclasts, where they’re involved in extracellular acidification and matrix destruction; however, the roles of their subunits in cancer cell proliferation, signaling, and other pro-tumor actions are not well established. Analysis of TCGA data shows that V-ATPase subunit Atp6v1c1 is overexpressed or amplified in 34% of human breast cancer cases, with a 2-fold decrease in survival at 12 years. Whereas other subunits, such as Atp6v1c2 and Atp6v0a3, are overexpressed or genomically amplified less often, 6% each respectively, and have less impact on survival. Experiments show that lentiviral-shRNA mediated ATP6v1c1 knockdown in 4T1 mouse mammary cancer cells significantly reduces orthotopic and intraosseous tumor growth. ATP6v1c1 knockdown also significantly reduces tumor stimulated bone resorption through osteoclastogenesis at the bone and metastasis in vivo, as well as V-ATPase activity, proliferation, and mTORC1 activation in vitro. To generalize the effects of ATP6v1c1 knockdown on proliferation and mTORC1 activation we used human cancer cell lines - MCF-7, MDA-MB-231, and MDA-MB-435s. ATP6V1C1 knockdown reduced cell proliferation and impaired mTORC1 pathway activation in cancer cells but not in the untransformed cell line C3H10T1/2. Our study reveals that V-ATPase activity may be mediated through mTORC1 and that ATP6v1c1 can be knocked down to block both V-ATPase and mTORC1 activity.