Spatial interplay of tissue hypoxia and T-cell regulation in ductal carcinoma in situ.

Hypoxia promotes aggressive tumor phenotypes and mediates the recruitment of suppressive T cells in invasive breast carcinomas. We investigated the role of hypoxia in relation to T-cell regulation in ductal carcinoma in situ (DCIS). We designed a deep learning system tailored for the tissue architecture complexity of DCIS, and compared pure DCIS cases with the synchronous DCIS and invasive components within invasive ductal carcinoma cases. Single-cell classification was applied in tandem with a new method for DCIS ductal segmentation in dual-stained CA9 and FOXP3, whole-tumor section digital pathology images. Pure DCIS typically has an intermediate level of colocalization of FOXP3+ and CA9+ cells, but in invasive carcinoma cases, the FOXP3+ (T-regulatory) cells may have relocated from the DCIS and into the invasive parts of the tumor, leading to high levels of colocalization in the invasive parts but low levels in the synchronous DCIS component. This may be due to invasive, hypoxic tumors evolving to recruit T-regulatory cells in order to evade immune predation. Our data support the notion that hypoxia promotes immune tolerance through recruitment of T-regulatory cells, and furthermore indicate a spatial pattern of relocalization of T-regulatory cells from DCIS to hypoxic tumor cells. Spatial colocalization of hypoxic and T-regulatory cells may be a key event and useful marker of DCIS progression.

Ductal Carcinoma in Situ: State-of-the-Art Review.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive cancer, and its detection, diagnosis, and management are controversial. DCIS incidence grew with the expansion of screening mammography programs in the 1980s and 1990s, and DCIS is viewed as a major driver of overdiagnosis and overtreatment. For pathologists, the diagnosis and classification of DCIS is challenging due to undersampling and interobserver variability. Understanding the progression from normal breast tissue to DCIS and, ultimately, to invasive cancer is limited by a paucity of natural history data with multiple proposed evolutionary models of DCIS initiation and progression. Although radiologists are familiar with the classic presentation of DCIS as asymptomatic calcifications at mammography, the expanded pool of modalities, advanced imaging techniques, and image analytics have identified multiple potential biomarkers of histopathologic characteristics and prognosis. Finally, there is growing interest in the nonsurgical management of DCIS, including active surveillance, to reduce overtreatment and provide patients with more personalized management options. However, current biomarkers are not adept at enabling identification of occult invasive disease at biopsy or accurately predicting the risk of progression to invasive disease. Several active surveillance trials are ongoing and are expected to better identify women with low-risk DCIS who may avoid surgery.

Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

Management of breast cancer in limited-resource settings is hindered by a lack of low-cost, logistically sustainable approaches toward molecular and cellular diagnostic pathology services that are needed to guide therapy. To address these limitations, we have developed a multimodal cellphone-based platform-the EpiView-D4-that can evaluate both cellular morphology and molecular expression of clinically relevant biomarkers directly from fine-needle aspiration (FNA) of breast tissue specimens within 1 h. The EpiView-D4 is comprised of two components: (1) an immunodiagnostic chip built upon a "non-fouling" polymer brush-coating (the "D4") which quantifies expression of protein biomarkers directly from crude cell lysates, and (2) a custom cellphone-based optical microscope ("EpiView") designed for imaging cytology preparations and D4 assay readout. As a proof-of-concept, we used the EpiView-D4 for assessment of human epidermal growth factor receptor-2 (HER2) expression and validated the performance using cancer cell lines, animal models, and human tissue specimens. We found that FNA cytology specimens (prepared in less than 5 min with rapid staining kits) imaged by the EpiView-D4 were adequate for assessment of lesional cellularity and tumor content. We also found our device could reliably distinguish between HER2 expression levels across multiple different cell lines and animal xenografts. In a pilot study with human tissue (n = 19), we were able to accurately categorize HER2-negative and HER2-positve tumors from FNA specimens. Taken together, the EpiView-D4 offers a promising alternative to invasive-and often unavailable-pathology services and may enable the democratization of effective breast cancer management in limited-resource settings.

Endometrial Adenocarcinomas With No Specific Molecular Profile: Morphologic Features and Molecular Alterations of "Copy-number Low" Tumors.

The study evaluated morphologic patterns, mutational profiles, and ß-catenin immunohistochemistry (IHC) in copy-number low (CNL) endometrial adenocarcinomas (EAs). CNL EAs (n=19) with next-generation or whole genome sequencing results and available tissue for IHC were identified from our institutional database. Clinical data and histologic slides were reviewed. IHC for ß-catenin was performed and correlated with mutation status. Images of digital slides of CNL EAs from The Cancer Genome Atlas (TCGA) database (n=90) were blindly reviewed by 4 pathologists, and morphology was correlated with mutation status. Categorical variables were analyzed using the Fisher exact test, and agreement was assessed using Fleiss κ. CTNNB1 mutations were present in 63% (12/19) of CNL EAs. ß-catenin nuclear localization was present in 83% of CTNNB1-mutated tumors (10/12) and in 0% (0/7) of CTNNB1-wildtype tumors (sensitivity 0.83, specificity 1.00). Squamous differentiation (SD) was present in 47% (9/19) and was more often observed in CTNNB1-mutated tumors (P=0.02). Mucinous differentiation (MD) was associated with KRAS mutations (P<0.01). Digital image review of TCGA CNL EAs revealed that pathologist agreement on SD was strong (κ=0.82), whereas agreement on MD was weak (κ=0.48). Pathologists identified SD in 22% (20/90), which was significantly associated with the presence of CTNNB1 mutations (P<0.01). CNL EAs demonstrate several morphologies with divergent molecular profiles. SD was significantly associated with CTNNB1 mutations and nuclear localization of ß-catenin in these tumors. Nuclear expression of ß-catenin is a sensitive and specific IHC marker for CTNNB1 mutations in CNL EAs. CNL EAs with KRAS mutations often displayed MD.

Unmasking the immune microecology of ductal carcinoma in situ with deep learning.

Despite increasing evidence supporting the clinical relevance of tumour infiltrating lymphocytes (TILs) in invasive breast cancer, TIL spatial variability within ductal carcinoma in situ (DCIS) samples and its association with progression are not well understood. To characterise tissue spatial architecture and the microenvironment of DCIS, we designed and validated a new deep learning pipeline, UNMaSk. Following automated detection of individual DCIS ducts using a new method IM-Net, we applied spatial tessellation to create virtual boundaries for each duct. To study local TIL infiltration for each duct, DRDIN was developed for mapping the distribution of TILs. In a dataset comprising grade 2-3 pure DCIS and DCIS adjacent to invasive cancer (adjacent DCIS), we found that pure DCIS cases had more TILs compared to adjacent DCIS. However, the colocalisation of TILs with DCIS ducts was significantly lower in pure DCIS compared to adjacent DCIS, which may suggest a more inflamed tissue ecology local to DCIS ducts in adjacent DCIS cases. Our study demonstrates that technological developments in deep convolutional neural networks and digital pathology can enable an automated morphological and microenvironmental analysis of DCIS, providing a new way to study differential immune ecology for individual ducts and identify new markers of progression.

Intra-tumor molecular heterogeneity in breast cancer: definitions of measures and association with distant recurrence-free survival.

PURPOSE: The purpose of the study was to define quantitative measures of intra-tumor heterogeneity in breast cancer based on histopathology data gathered from multiple samples on individual patients and determine their association with distant recurrence-free survival (DRFS). METHODS: We collected data from 971 invasive breast cancers, from 1st January 2000 to 23rd March 2014, that underwent repeat tumor sampling at our institution. We defined and calculated 31 measures of intra-tumor heterogeneity including ER, PR, and HER2 immunohistochemistry (IHC), proliferation, EGFR IHC, grade, and histology. For each heterogeneity measure, Cox proportional hazards models were used to determine whether patients with heterogeneous disease had different distant recurrence-free survival (DRFS) than those with homogeneous disease. RESULTS: The presence of heterogeneity in ER percentage staining was prognostic of reduced DRFS with a hazard ratio of 4.26 (95% CI 2.22-8.18, p < 0.00002). It remained significant after controlling for the ER status itself (p < 0.00062) and for patients that had chemotherapy (p < 0.00032). Most of the heterogeneity measures did not show any association with DRFS despite the considerable sample size. CONCLUSIONS: Intra-tumor heterogeneity of ER receptor status may be a predictor of patient DRFS. Histopathologic data from multiple tissue samples may offer a view of tumor heterogeneity and assess recurrence risk.

Prospective Evaluation of Lymph Node Processing at Staging Surgery for High-grade Endometrial Cancer.

To determine whether the processing of additional adipose tissue collected during lymph node (LN) dissection results in the identification of additional LNs during endometrial cancer (EC) staging and to determine if the division of LNs into nodal basin-specific specimens has an effect on the number of LNs identified during EC staging. A prospective randomized controlled trial was performed on women with high-grade EC undergoing surgical staging. Subjects were randomized to collection of LNs into nodal basin-specific containers on the randomized side versus simple labeling on the nonrandomized side. The total number of LNs and total number of LNs with metastases on the randomized versus the nonrandomized side were compared. The remaining adipose tissue from each LN specimen was submitted for histologic examination. We analyzed the number of LNs with and without metastases identified from additional adipose tissue. Of 120 consented subjects, 56 had sufficient data for analysis. The additional adipose tissue contained 7.5 additional LNs per patient on average (range: 0-26). In 2/54 total cases (3.7%) and 2/5 cases with nodal metastases (40%), the additional adipose contained LNs with metastases. In both cases, metastases were also detected in grossly identified LN candidates. The mean number of LNs identified was not significantly different based on method of collection (P=0.22). The mean number of LNs containing metastases per side was not significantly different (P=0.58). Processing of adipose tissue does increase the total number of LNs identified, however, it does not influence EC stage. No difference in LN counts was noted with basin-specific collection.

Maternal CD4+ T cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission.

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4(+) T-cell depleted at the time of inoculation. Animals that received the CD4(+) T-cell-depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8(+) T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4(+) T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.

Cystic fibrosis involving the cervix, mimicking a well-differentiated adenocarcinoma: a case report.

We describe clinicopathologic and immunohistochemical features of an unusual case of cystic fibrosis manifesting in the cervix as a mass lesion, mimicking cervical adenocarcinoma. A 24-year-old nulligravida with cystic fibrosis developed heavy postcoital vaginal bleeding 4 months after starting oral contraceptives and was found to have a cervical mass. She underwent a loop electrosurgical excision of the mass, and microscopic examination revealed a florid endocervical proliferation, extending to the margins. This lesion was initially interpreted as an invasive, well-differentiated endocervical adenocarcinoma. However, on subsequent review, the lesion was found to have a low rate of proliferation, no evidence of an infiltrative growth pattern, and abundant acute inflammation. Given these findings and the absence of any residual endocervical lesion on a subsequent cold knife conization, we determined that this was a benign, likely reactive, lesion. This case, together with previous studies, suggests that women with cystic fibrosis can develop proliferative endocervical lesions and that oral contraceptives may contribute to their development.

Genes with bimodal expression are robust diagnostic targets that define distinct subtypes of epithelial ovarian cancer with different overall survival.

In some cancer types, certain genes behave as molecular switches, with on and off expression states. These genes tend to define tumor subtypes associated with different treatments and different patient survival. We hypothesized that clinically relevant molecular switch genes exist in epithelial ovarian cancer. To test this hypothesis, we applied a bimodal discovery algorithm to a publicly available ovarian cancer expression microarray data set, GSE9891 [285 tumors: 246 malignant serous (MS), 20 endometrioid (EM), and 18 low malignant potential (LMP) ovarian carcinomas]. Genes with robust bimodal expression patterns were identified across all ovarian tumor types and also within selected subtypes: 73 bimodal genes demonstrated differential expression between LMP versus MS and EM; 22 bimodal genes distinguished MS from EM; and 14 genes had significant association with survival among MS tumors. When these genes were combined into a single survival score, the median survival for patients with a favorable versus unfavorable score was 65 versus 29 months (P < 0.0001, hazard ratio = 0.4221). Two independent data sets [high-grade, advanced-stage serous (n = 53) and advanced-stage (n = 119) ovarian tumors] validated the survival score performance. We conclude that genes with bimodal expression patterns not only define clinically relevant molecular subtypes of ovarian carcinoma but also provide ideal targets for translation into the clinical laboratory.

High potency silencing by single-stranded boranophosphate siRNA.

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.

RNA interference using boranophosphate siRNAs: structure-activity relationships.

In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.

RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells.

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.