In situ HER2 RNA expression as a predictor of pathologic complete response of HER2-positive breast cancer patients receiving neoadjuvant chemotherapy and anti-HER2 targeted treatment.

BACKGROUND: Immunohistochemistry (IHC) and in situ hybridization (ISH) remain standard biomarkers for therapeutic decisions in human epidermal growth factor 2 (HER2)-positive breast cancers (BCs); however, they are insufficient to explain the heterogeneous anti-HER2 response. METHODS: We aimed to investigate the correlation of in situ HER2 RNA expression (isHRE), using RNAscope, with HER2 biomarkers and the impact of isHRE on the pathological complete response (pCR) rates of 278 patients with HER2 IHC/fluorescence ISH (FISH)-positive BC receiving neoadjuvant chemotherapy and anti-HER2 targeted treatment (NCTT). RESULTS: We validated HER2 RNAscope scoring as a semiquantitative method to determine isHRE and showed a positive correlation between RNAscope scores and pCR rates, with particularly different rates between patients with a score of 5 versus 1-4 BCs (66.7% vs. 15.9%, p < 0.0001). There were higher RNAscope scores and pCR rates in patients with HER2 IHC 3 + versus IHC 2+/FISH + BCs and HER2 RNAscope scores and pCR rates showed similar non-linear positive correlations with HER2 copy numbers and HER2/centromere 17 ratios. Moreover, in each HER2-positive IHC/FISH category, higher pCR rates were observed in patients with RNAscope scores of 5 versus 1-4 BC. Patients achieving pCR had BCs with notably higher HER2 RNAscope scores. Multivariate analysis identified HER2 RNAscope 5 as a strong pCR predictor [odds ratio = 10.865, p < 0.001]. The combined impact of multivariate analysis-defined pCR predictors demonstrated that a higher pCR rate was observed in patients with a score of 5 versus a score of 1-4 BCs regardless of the status of hormone receptor and mono-or dual anti-HER2 blockade. CONCUSIONS: Our results demonstrated that high isHRE (RNAscope score 5) is a strong pCR predictor in patients with HER2-positive BCs receiving NCTT, highlighting the complementary role of isHRE in stratifying HER2 status in tissue. Such stratification is relevant to anti-HER2 therapeutic efficacy, particularly using the cutoff of score 1-4 versus 5.

Transcriptomic alterations underlying metaplasia into specific metaplastic components in metaplastic breast carcinoma.

BACKGROUND: Metaplastic breast carcinoma (MpBC) typically consists of carcinoma of no special type (NST) with various metaplastic components. Although previous transcriptomic and proteomic studies have reported subtype-related heterogeneity, the intracase transcriptomic alterations between metaplastic components and paired NST components, which are critical for understanding the pathogenesis underlying the metaplastic processes, remain unclear. METHODS: Fifty-nine NST components and paired metaplastic components (spindle carcinomatous [SPS], matrix-producing, rhabdoid [RHA], and squamous carcinomatous [SQC] components) were microdissected from specimens obtained from 27 patients with MpBC for gene expression profiling using the NanoString Breast Cancer 360 Panel on a NanoString nCounter FLEX platform. BC360-defined signatures were scored using nSolver software. RESULTS: Hierarchical clustering and principal component analysis revealed a heterogeneous gene expression profile (GEP) corresponding to the NST components, but the GEP of metaplastic components exhibited subtype dependence. Compared with the paired NST components, the SPS components demonstrated the upregulation of genes related to stem cells and epithelial-mesenchymal transition and displayed enrichment in claudin-low and macrophage signatures. Despite certain overlaps in the enriched functions and signatures between the RHA and SPS components, the specific differentially expressed genes differed. We observed the RHA-specific upregulation of genes associated with vascular endothelial growth factor signaling. The chondroid matrix-producing components demonstrated the upregulation of hypoxia-related genes and the downregulation of the immune-related MHC2 signature and the TIGIT gene. In the SQC components, TGF-ß and genes associated with cell adhesion were upregulated. The differentially expressed genes among metaplastic components in the 22 MpBC cases with one or predominantly one metaplastic component clustered paired NST samples into clusters with correlation with their associated metaplastic types. These genes could be used to separate the 31 metaplastic components according to respective metaplastic types with an accuracy of 74.2%, suggesting that intrinsic signatures of NST may determine paired metaplastic type. Finally, the EMT activity and stem cell traits in the NST components were correlated with specimens displaying lymph node metastasis. CONCLUSIONS: We presented the distinct transcriptomic alterations underlying metaplasia into specific metaplastic components in MpBCs, which contributes to the understanding of the pathogenesis underlying morphologically distinct metaplasia in MpBCs.

Clinical Outcomes of Metastatic Breast Cancer in Patients Having Imaging Liver Pseudocirrhosis with or without Evident Varices.

BACKGROUND: Pseudocirrhosis is an imaging finding of malignancies with liver metastasis with or without clinical liver cirrhosis-related portal hypertension (pHTN). This study defined evident pHTN by the presence of esophageal or gastric varices and compared patients' outcomes of metastatic breast cancer with imaging-diagnosed pseudocirrhosis with or without varices. METHODS: The medical records from patients with metastatic breast cancer and pseudocirrhosis between 2005 and 2017 were retrospectively analyzed. Survival outcomes were compared based on endoscopic evidence of esophageal or gastric varices. RESULTS: Among 106 patients with pseudocirrhosis, 33 (31%) had de novo stage IV disease, and 66 (62%) had hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Eighty-one (76%) had initial metastases in both hepatic lobes, and 32 (30%) had esophageal or gastric varices. The median overall survival (OS) was 5 and 13 months in patients with and without varices (P = .002). The median OS in patients with HER2-positive, HR-positive/HER2-negative, and triple-negative subtype was 16, 9, and 2 months, respectively (P = .001). Patients with varices usually had cirrhotic complications, including gastrointestinal bleeding, hyperbilirubinemia, hyperammonemia, and coagulopathy. Despite their challenging clinical conditions, 7 patients with varices had OS exceeding 1 year. In multivariate analysis, evident varices (P = .007) and triple-negative subtype (P = .013) were associated with poor OS. CONCLUSIONS: Patients with pseudocirrhosis and evident varices had a significantly shorter median OS, and were usually associated with clinical cirrhosis-related complications. To maximize OS, early identification and meticulous supportive care are warranted.

Epibulbar complex choristoma with simultaneous involvement of eyelid: a case report.

BACKGROUND: Epibulbar complex choristoma, a rare congenital epibulbar tumor, has many diverse forms. Reviewing the literature, it can present clinically as either a circumferential or isolated epibulbar mass, limbal tumor, lateral canthal mass, aggregate of ectopic cilia in the upper eyelid, eyelid mass mimicking chalazion, or lacrimal caruncle mass. The management depends on the extent of involvement, the risk of amblyopia, and cosmetic concerns. Here, we report an atypical presentation of epibulbar complex choristoma with simultaneous eyelid involvement. CASE PRESENTATION: A 1-month-old full-term boy was brought to our clinic with congenital epibulbar mass of the right eye with simultaneous eyelid involvement. Dilated fundus examination was unremarkable. Survey for linear nevus sebaceous Jadassohn was negative. Due to concerns of possible amblyopia and cosmetics, lamellar keratectomy, sclerotomy, and conjunctivoplasty were performed to remove the epibulbar lesion. The eyelid defect was reconstructed with 6-0 Vicryl sutures. Histopathological examination reported complex choristoma. Upon three-year follow-up, low astigmatism and favorable cosmetics results were achieved. CONCLUSIONS: Congenital complex choristoma can present clinically as an epibulbar mass with eyelid involvement. The management depends on the extent of involvement, the risk of amblyopia, and cosmetic concerns. The method of eyelid reconstruction should be tailored according to the residual eyelid defect.

The breast graded prognostic assessment is associated with the survival outcomes in breast cancer patients receiving whole brain re-irradiation.

INTRODUCTION: Whole brain (WB) re-irradiation for breast cancer patients with progressive brain metastasis after first-course WB radiotherapy (WBRT) is controversial. In this study, we sought to investigate the association between the molecular sub-classifications and breast-specific Graded Prognostic Assessment (GPA, which includes the Karnofsky performance status, molecular subtypes, and age as its indices) and the outcomes of breast cancer patients who received WB re-irradiation. METHODS: Twenty-three breast cancer patients who received WB re-irradiation for relapsed and progressive intracranial lesions after first-course WBRT between 2004 and 2016 were retrospectively reviewed. Patients were divided according to the 4 molecular subtypes of luminal A/B (hormone receptor [HR]+/human epidermal growth factor receptor 2 [HER2]-), luminal HER2 (HR+/HER2+), HER2 (HR-/HER2+), and triple negative (HR-/HER2-). The clinical and radiological responses and survival rates after WB re-irradiation were analyzed. RESULTS: At 1 month after WB re-irradiation, 13 of 23 patients (56.5%) exhibited disappearance or alleviation of neurological symptoms. The median survival time after WB re-irradiation was 2.93 months (95% confidence interval [CI], 1.79-4.08). After WB re-irradiation, patients with HER2-negative tumors had poorer median survival times than those with HER2-positive tumors (2.23 vs. 3.0 months, respectively; p = 0.022). Furthermore, patients with high breast GPA scores (2.5-4.0, n = 11) had longer median survivals than those with low-scores (0-2.0, n = 12) after WB re-irradiation (4.37 vs. 1.57 months, respectively; p < 0.005). CONCLUSIONS: WB re-irradiation may be a feasible treatment option for certain breast cancer patients who develop brain metastatic lesions after first-course WBRT when these lesions are ineligible for radiosurgery or surgery.

PRRX2 as a novel TGF-ß-induced factor enhances invasion and migration in mammary epithelial cell and correlates with poor prognosis in breast cancer.

TGF-ß and cancer progression share a multifaceted relationship. Despite the knowledge of TGF-ß biology in the development of cancer, several factors that mediate the cancer-promoting role of TGF-ß continue to be identified. This study aimed to identify and characterise novel factors potentially related to TGF-ß-mediated tumour aggression in breast cells. We treated the human mammary epithelial cell line MCF10A with TGF-ß and identified TGF-ß-dependent upregulation of PRRX2, the gene encoding paired-related homeobox 2 transcription factor. Overexpression of PRRX2 enhanced migration, invasion and anchorage-independent growth of MCF10A cells and induced partial epithelial mesenchymal transition (EMT), as determined by partial fibroblastoid morphology of cells, upregulation of EMT markers and partially disrupted acinar structure in a three-dimensional culture. We further identified PLAT, the gene encoding tissue-type plasminogen activator (tPA), as the highest differentially expressed gene in PRRX2-overexpressing MCF10A cells, and demonstrated direct binding and transactivation of the PLAT promoter by PRRX2. Furthermore, PLAT knockdown inhibited PRRX2-mediated enhanced migration and invasion, suggesting that tPA may mediate PRRX2-induced migration and invasion. Finally, the significant correlation of PRRX2 expression with poor survival in 118 primary breast tumour samples (P = 0.027) and the increased PRRX2 expression in metaplastic breast carcinoma samples, which is pathogenetically related to EMT, validated the biological importance of PRRX2-enhanced migration and invasion and PRRX2-induced EMT. Thus, our data suggest that upregulation of PRRX2 may be a mechanism contributing to TGF-ß-induced invasion and EMT in breast cancer. © 2016 Wiley Periodicals, Inc.

Locoregional Recurrence Risk for Postmastectomy Breast Cancer Patients With T1-2 and One to Three Positive Lymph Nodes Receiving Modern Systemic Treatment Without Radiotherapy.

BACKGROUND: Administering postmastectomy radiotherapy (PMRT) to patients with T1-2 breast cancer and one to three positive axillary lymph nodes (ALNs) is controversial. The current study assessed the association of clinicopathologic features and molecular subclassification with locoregional recurrence (LRR) in patients who did not receive PMRT. METHODS: Between January 2004 and December 2008, 293 patients with T1-2 breast cancer and one to three positive ALNs not receiving PMRT were analyzed. Most of the patients received an anthracycline- or taxane-based regimen or both. The patients were divided according to the four molecular subtypes as follows: luminal A/B, luminal human epidermal growth factor receptor 2 (HER2), HER2, and triple-negative breast cancer. Overall survival (OS) and LRR were calculated using the Kaplan-Meier method, and the clinicopathologic prognostic factors were compared using log-rank tests and the Cox regression model. RESULTS: After a median follow-up period of 82.8 months, the 10-year LRR and OS were respectively 10 %, and 88.9 %. The patients with triple-negative breast cancer had a higher 5-year LRR rate (10.6 %) than those without this disease (4.2 %) (p = 0.05). Multivariate analysis showed that young age (≤40 years), tumor larger than 3 cm, and the presence of extensive intraductal components were significant risk factors for LRR. The 5-year LRR was 3.1 % for the patients without the aforementioned risk factors, 7.9 % for those with one risk factor, and 25 % for those with two or more risk factors (p < 0.001). CONCLUSIONS: Administering modern systemic therapy to early breast cancer patients not receiving PMRT reduced the LRR rate. Younger patients, those with a tumor larger than 3 cm, and those with extensive intraductal components might benefit from PMRT.

Mutational analysis of MED12 exon 2 in a spectrum of fibroepithelial tumours of the breast: implications for pathogenesis and histogenesis.

AIMS: Fibroadenomas (FAs) and phyllodes tumours (PTs) are fibroepithelial tumours. Mutations in MED12 exon 2 have been reported in FAs. This study investigated the MED12 mutations in a spectrum of fibroepithelial tumours. METHODS AND RESULTS: Using direct sequencing, we analysed MED12 exon 2 mutations on 121 samples, including PTs and FAs and variants. We found MED12 mutations in 71.4% of PTs. No significant difference in the mutation frequency was observed between benign, borderline and malignant PTs, and a general lack of correlation existed between mutations and pathological factors associated with PT grading. The mutation patterns were similar between PTs and FAs, with codon 44 being involved most frequently. MED12 mutations were identified in 47.1, 52.6 and 50.0% of complex FAs, juvenile FAs and tubular adenomas (TAs), respectively, and the frequency and mutation patterns were similar between these FA variants and usual FAs. CONCLUSIONS: The high frequency and similar patterns of MED12 mutations in FAs and various grades of PTs implies that the MED12 mutation is a common and early pathological event in these fibroepithelial tumours. The similar frequency and patterns of the MED12 mutation between FAs and variants suggests that FA variants are bona fide FAs, with identical pathogenesis involving MED12 mutations.

Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast.

Mutations in HER2 gene have been identified in a small subset of breast cancer cases. Identification of HER2 mutation has therapeutic implications for breast cancer, but whether a subgroup of breast cancer with a higher frequency of HER2 mutation exists, remains unknown. We analyzed HER2 mutation and pathologic factors on 73 formalin-fixed, paraffin-embedded samples, including 21 pleomorphic invasive lobular carcinoma (p-ILC) cases, 3 pleomorphic lobular carcinoma in situ (p-LCIS) cases, and 49 classic invasive lobular carcinoma (c-ILC) cases. Mutations were identified through direct sequencing. HER2 overexpression and amplification were determined through immunohistochemistry and fluorescent in situ hybridization. Six mutations were identified, including five in the 24 p-ILC or p-LCIS (p-ILC/p-LCIS) cases (20.8 %) and one in the 49 c-ILC cases (2.0 %), and the difference in frequency was significant (p = 0.013). Eight of the 24 (33.3 %) p-ILC/p-LCIS cases exhibited HER2 amplification or overexpression (amplification/overexpression), which was significantly higher than in the c-ILC cases (1/49, 2 %). Mutation and amplification/overexpression were mutually exclusive. HER2 mutations were identified more frequently in the p-ILC/p-LCIS cases with extensive apocrine change (p = 0.018). Combined HER2 alterations through mutation or amplification/overexpression were more frequently identified in p-ILC/p-LCIS cases without estrogen receptor expression. The high frequency (54.1 %, 13/24) of combined HER2 alterations in the p-ILC/p-LCIS cases suggests a crucial role of HER2 in the pathogenesis of p-ILC/p-LCIS. Because of the reported responsiveness of HER2 mutation to anti-HER2 therapy, p-ILC patients without HER2 amplification/overexpression should receive HER2 mutation analysis to identify this therapeutically relevant target.

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells.

INTRODUCTION: Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. METHODS: Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. RESULTS: Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH+/GD2+ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH+ or ALDH+/GD2+ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes. CONCLUSIONS: Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH+ and ALDH+/GD2+ subpopulations.

Differential expression of hyaluronan synthase 2 in breast carcinoma and its biological significance.

AIMS: Hyaluronan synthase 2 (HAS2) is an enzyme in hyaluronan synthesis. Several studies have demonstrated that HAS2 plays a critical role in tumour progression in breast cancer cells. The in-situ expression patterns of HAS2 remain unclear, and the aim of this study was to determine these in order to elucidate the role of HAS2 in breast cancer. METHODS AND RESULTS: We examined HAS2 expression using immunohistochemistry in 244 breast carcinomas of various subtypes. We found expression of HAS2 in 30.6% of invasive ductal carcinomas (IDCs); in IDCs, HAS2 expression was correlated significantly with the triple-negative phenotype and the basal-like phenotype, and univariate and multivariate analyses indicated that it was associated with poorer overall survival. In contrast to other carcinoma subtypes, HAS2 expression was observed in up to 72.7% of metaplastic carcinomas of breast (MCB), a carcinoma subtype related to the epithelial-mesenchymal transition (EMT). Consistently, we noted up-regulated levels of HAS2 RNA and protein in TGF-ß-induced EMT in MCF-10A mammary epithelial cells. CONCLUSION: Our findings demonstrate that HAS2 plays a role in aggressive phenotypes of primary breast carcinoma. The strong expression of HAS2 in MCB and the up-regulation of HAS2 in breast cells induced to exhibit EMT implicates an association between HAS2 expression and EMT in breast cancer.

Correlation of In Situ HER2 RNA Expression With HER2 Immunohistochemistry and Fluorescence In Situ Hybridization Categories in Breast Cancer.

CONTEXT.­: RNA sequencing study has demonstrated that human epidermal growth factor receptor 2 (HER2) RNA levels influence anti-HER2 therapeutic efficacy. However, in situ HER2 RNA expression (isHRE), which evaluates HER2 RNA expression in tissue, has remained unclear in breast cancers (BCs) of various HER2 immunohistochemistry (IHC)/in situ hybridization (ISH) categories. OBJECTIVE.­: To correlate isHRE with all HER2 IHC/fluorescence ISH (FISH) categories in BC. DESIGN.­: Formalin-fixed, paraffin-embedded tissue sections from 259 BCs, covering all IHC/FISH categories, were analyzed for isHRE by RNAscope. RESULTS.­: We validated HER2 RNAscope scoring as a semiquantitative method to evaluate isHRE and demonstrated significantly higher RNAscope scores in IHC 3+ than in IHC 2+ cases, and in IHC 2+ than in IHC 0/1+ cases. Among the 5 IHC 2+/FISH groups, group 1 (G1) cases had the highest scores. The scores in G3 cases were higher than those in G2, but not significantly different from those in G4 and G5. G4 cases had significantly higher scores than those in G2. Higher HER2 copy numbers and HER2:CEP 17 (centromere 17) copy number ratios were significantly correlated with higher isHRE in G1 cases, but not in G2 to G5 cases. RNAscope scores were significantly lower in HER2-negative (IHC 0) than in HER2-low (IHC 2+/FISH- and IHC 1+) BCs but were not different between IHC 0 and 1+ BCs when analyzed separately. CONCLUSIONS.­: We demonstrate the HER2 RNA expression status among BCs of various HER2 IHC/FISH categories in tissue. Such information may be relevant for anti-HER2 treatment decisions considering the role of HER2 RNA expression in predicting anti-HER2 therapeutic efficacy.

Tonsil tissue control is ideal for monitoring estrogen receptor immunohistochemical staining.

BACKGROUND: Estrogen receptor (ER) testing performed using immunohistochemistry (IHC) is a critical predictive tool for breast cancer treatment. This study aimed to investigate the use of tonsil control for monitoring ER staining and hypothesize that optimal staining would reduce interlaboratory variations. METHODS: A proficiency test for ER IHC was conducted using 21 tissue cores. The staining quality was centrally reviewed based on tonsil ER staining. RESULTS: We found that 64.9% of participant samples demonstrated optimal or good staining quality. Poor staining quality was significantly associated with the use of Ventana autostainers and concentrated antibodies. Although the concordance rate did not show significant differences across staining quality levels, interparticipant agreement declined as staining quality deteriorated. Among the 19 discordant responses, 63.2% could be attributed to staining problems, whereas 36.8% could be due to misinterpretation. Poor staining quality due to inadequate staining was the primary reason for undercalls, which can lead to false-negative results. Misinterpretations of nonspecific faint staining that was weaker than the staining of the tonsil control were the cause of most overcalls. CONCLUSION: Tonsil tissue is an ideal control for monitoring ER staining and can serve as a reference for determining the lower bound for ER positivity. Optimal ER staining and appropriate references for ER positivity can further improve ER IHC quality.

Differential expression of moesin in breast cancers and its implication in epithelial-mesenchymal transition.

AIMS: Moesin belongs to the ERM (ezrin, radixin and moesin) family. Recent in-vitro studies have shown the possible involvement of moesin in epithelial-mesenchymal transition (EMT), but correlating in-vivo evidence is lacking. METHODS AND RESULTS: To study the biological significance of moesin, we used immunohistochemistry to investigate the in-situ expression profiles of moesin in 322 breast carcinomas of different subtypes, including 23 cases of metaplastic carcinoma (MCB) which is pathogenetically considered to involve EMT. Moesin was highly expressed in 95.7% of cases of MCB, and in 16% of cases of invasive ductal carcinoma (IDC), but was negative in all other subtypes of breast carcinomas. In IDCs, moesin expression correlated positively with a high histological grade (P < 0.001), basal-like phenotype (P < 0.001) and poor overall survival (P = 0.0263). Transfection of MCF7 cells with Snail, one of the key regulators of EMT, showed up-regulation of moesin at the transcriptional level. Finally, mRNA level of moesin correlated positively with Snail and EMT-related genes in a microarray data set using primary breast cancer samples. CONCLUSION: These results offer biological evidence of moesin as an EMT marker, support the association between moesin, Snail and EMT and suggest a role for moesin in breast cancer prognostication.

Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2.

Pathological expression of human ErbB-2 protein, also known as HER-2, is common in many types of cancer. ErbB-2 is a member of the EGF receptor tyrosine kinase family and has been rigorously studied as a signaling molecule on the cell membrane. Here, we report that ErbB-2 is also expressed in the nucleus in cultured cells as well as primary tumor tissues. Nuclear ErbB-2 was found to associate with multiple genomic targets in vivo, including the cyclooxygenase enzyme COX-2 gene promoter. ErbB-2 forms a complex at a specific nucleotide sequence of the COX-2 promoter and is able to stimulate its transcription. This study demonstrates the presence of ErbB-2 in the nucleus and identifies the function of ErbB-2 as a transcriptional regulator.

Human herpesvirus 8-associated lymphoma mimicking cutaneous anaplastic large T-cell lymphoma in a patient with human immunodeficiency virus infection.

Primary effusion lymphoma, a human herpesvirus 8 (HHV8)-associated lymphoma, is uncommon, and it is usually seen in human immunodeficiency virus (HIV)-infected patients. It presents as a body cavity-based lymphomatous effusion, but several cases of the so-called solid primary effusion lymphoma presenting as solid tumors without associated lymphomatous effusion have been reported. They have similar clinical, histopathological and immunophenotypical features. Most of them have a B-cell genotype. This suggests the solid variant may represent a clinicopathological spectrum of primary effusion lymphoma. We report a case of HHV8-associated lymphoma histopathologically and immunophenotypically mimicking cutaneous anaplastic large cell lymphoma. The patient was a 31-year-old HIV-seropositive man presenting with skin nodules over his right thigh. Biopsy of the nodules showed anaplastic large cells infiltrating the dermis. These malignant cells strongly expressed CD3, CD30 and CD43. Cutaneous anaplastic large T-cell lymphoma was initially diagnosed, but further tests, including immunoreactivity for HHV8 protein and clonal rearrangements of immunoglobulin genes, confirmed the diagnosis of HHV8-associated B-cell lymphoma with aberrant T-cell marker expression. This case provides an example of solid primary effusion lymphoma mimicking cutaneous anaplastic large T-cell lymphoma and highlights the importance of HHV8 immunohistochemistry and molecular tests in the diagnosis of HHV8-associated lymphoma with a cutaneous presentation.

Fast Segmentation of Metastatic Foci in H&E Whole-Slide Images for Breast Cancer Diagnosis.

Breast cancer is the leading cause of death for women globally. In clinical practice, pathologists visually scan over enormous amounts of gigapixel microscopic tissue slide images, which is a tedious and challenging task. In breast cancer diagnosis, micro-metastases and especially isolated tumor cells are extremely difficult to detect and are easily neglected because tiny metastatic foci might be missed in visual examinations by medical doctors. However, the literature poorly explores the detection of isolated tumor cells, which could be recognized as a viable marker to determine the prognosis for T1NoMo breast cancer patients. To address these issues, we present a deep learning-based framework for efficient and robust lymph node metastasis segmentation in routinely used histopathological hematoxylin−eosin-stained (H−E) whole-slide images (WSI) in minutes, and a quantitative evaluation is conducted using 188 WSIs, containing 94 pairs of H−E-stained WSIs and immunohistochemical CK(AE1/AE3)-stained WSIs, which are used to produce a reliable and objective reference standard. The quantitative results demonstrate that the proposed method achieves 89.6% precision, 83.8% recall, 84.4% F1-score, and 74.9% mIoU, and that it performs significantly better than eight deep learning approaches, including two recently published models (v3_DCNN and Xception-65), and three variants of Deeplabv3+ with three different backbones, namely, U-Net, SegNet, and FCN, in precision, recall, F1-score, and mIoU (p<0.001). Importantly, the proposed system is shown to be capable of identifying tiny metastatic foci in challenging cases, for which there are high probabilities of misdiagnosis in visual inspection, while the baseline approaches tend to fail in detecting tiny metastatic foci. For computational time comparison, the proposed method takes 2.4 min for processing a WSI utilizing four NVIDIA Geforce GTX 1080Ti GPU cards and 9.6 min using a single NVIDIA Geforce GTX 1080Ti GPU card, and is notably faster than the baseline methods (4-times faster than U-Net and SegNet, 5-times faster than FCN, 2-times faster than the 3 different variants of Deeplabv3+, 1.4-times faster than v3_DCNN, and 41-times faster than Xception-65).

Fast cross-staining alignment of gigapixel whole slide images with application to prostate cancer and breast cancer analysis.

Joint analysis of multiple protein expressions and tissue morphology patterns is important for disease diagnosis, treatment planning, and drug development, requiring cross-staining alignment of multiple immunohistochemical and histopathological slides. However, cross-staining alignment of enormous gigapixel whole slide images (WSIs) at single cell precision is difficult. Apart from gigantic data dimensions of WSIs, there are large variations on the cell appearance and tissue morphology across different staining together with morphological deformations caused by slide preparation. The goal of this study is to build an image registration framework for cross-staining alignment of gigapixel WSIs of histopathological and immunohistochemical microscopic slides and assess its clinical applicability. To the authors' best knowledge, this is the first study to perform real time fully automatic cross staining alignment of WSIs with 40× and 20× objective magnification. The proposed WSI registration framework consists of a rapid global image registration module, a real time interactive field of view (FOV) localization model and a real time propagated multi-level image registration module. In this study, the proposed method is evaluated on two kinds of cancer datasets from two hospitals using different digital scanners, including a dual staining breast cancer data set with 43 hematoxylin and eosin (H&E) WSIs and 43 immunohistochemical (IHC) CK(AE1/AE3) WSIs, and a triple staining prostate cancer data set containing 30 H&E WSIs, 30 IHC CK18 WSIs, and 30 IHC HMCK WSIs. In evaluation, the registration performance is measured by not only registration accuracy but also computational time. The results show that the proposed method achieves high accuracy of 0.833 ± 0.0674 for the triple-staining prostate cancer data set and 0.931 ± 0.0455 for the dual-staining breast cancer data set, respectively, and takes only 4.34 s per WSI registration on average. In addition, for 30.23% data, the proposed method takes less than 1 s for WSI registration. In comparison with the benchmark methods, the proposed method demonstrates superior performance in registration accuracy and computational time, which has great potentials for assisting medical doctors to identify cancerous tissues and determine the cancer stage in clinical practice.

Tumor-infiltrating lymphocyte abundance and programmed death-ligand 1 expression in metaplastic breast carcinoma: implications for distinct immune microenvironments in different metaplastic components.

Both stromal tumor-infiltrating lymphocytes (sTILs) and programmed death-ligand 1 (PD-L1) affect responses to immunotherapy; however, the extent of sTIL and PD-L1 expression within various metaplastic components in metaplastic breast carcinoma (MBC), which are critical for the characterization of immune microenvironments, remains unreported. We profiled sTIL infiltration and PD-L1 expression in different metaplastic components of specimens from 82 MBC patients. The overall positivity for high or intermediate (H/I) sTIL, immune cell-PD-L1 (IcPD-L1), and tumor cell-PD-L1 (TcPD-L1) was 34.1%, 47.6%, and 17.1%, respectively, but differences specific to MBC subtypes and each metaplastic component existed. Squamous cell carcinoma exhibited the highest positivity rates of sTIL(H/I) (50.0%) and IcPD-L1 (66.7%), while matrix-producing carcinoma had the lowest respective rates (14.3% and 28.6%). The positivity rates of sTIL(H/I) and IcPD-L1 were the highest in squamous component (Sq) and the lowest in chondroid component (Ch). All cases that had discordant sTIL categories between carcinoma of no special type (NST) and metaplastic components showed sTIL(H/I) positivity higher in Sq, but lower in spindled component (Sp) and Ch. While there was no pattern of higher IcPD-L1-positivity in Sp, six of the seven cases that were TcPD-L1-discordant between NST and Sp were TcPD-L1-positive in Sp, suggesting a trend for higher TcPD-L1 in Sp. The diagnostic predictability of total tumor IcPD-L1 positivity based on IcPD-L1 positivity in Sq and Ch was 95.2% and 33.3%, respectively. Multivariate analysis showed that sTIL(H/I) positivity, but not PD-L1 positivity, correlated with better survival. Our data implicate distinct immune microenvironments in different metaplastic components in MBC, which may have immunopathologic, diagnostic, and therapeutic significance.