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Background Elevated mammographic breast density is a strong breast cancer risk factor with poorly understood etiology. Increased deposition of collagen, one of the main fibrous proteins present in breast stroma, has been associated with increased mammographic density. Collagen fiber architecture has been linked to poor outcomes in breast cancer. However, relationships of quantitative collagen fiber features assessed in diagnostic biopsies with mammographic density and lesion severity are not well-established. Methods Clinically indicated breast biopsies from 65 in situ or invasive breast cancer cases and 73 frequency matched-controls with a benign biopsy result were used to measure collagen fiber features (length, straightness, width, alignment, orientation and density (fibers/µm2)) using second harmonic generation microscopy in up to three regions of interest (ROIs) per biopsy: normal, benign breast disease, and cancer. Local and global mammographic density volumes were quantified in the ipsilateral breast in pre-biopsy full-field digital mammograms. Associations of fibrillar collagen features with mammographic density and severity of biopsy diagnosis were evaluated using generalized estimating equation models with an independent correlation structure to account for multiple ROIs within each biopsy section. Results Collagen fiber density was positively associated with the proportion of stroma on the biopsy slide (p p = 0.035) and within a 2 mm perilesional ring (p = 0.02), but not with global mammographic density measures. As severity of the breast biopsy diagnosis increased at the ROI level, collagen fibers tended to be less dense, shorter, straighter, thinner, and more aligned with one another (p Conclusions Collagen fiber density was positively associated with local, but not global, mammographic density, suggesting that collagen microarchitecture may not translate into macroscopic mammographic features. However, collagen fiber features may be markers of cancer risk and/or progression among women referred for biopsy based on abnormal breast imaging.

Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging.Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63).IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 μg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P.0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P.0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis.This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC., Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC) and the mechanisms driving benefit are poorly understood. Here, the authors show that elevated Notch signaling predicts clinical benefit in ICB in relapsed SCLC.

We present a case of a 69-year-old male patient diagnosed with high grade (T1 HG) urothelial carcinoma of the bladder who progressed rapidly towards muscle invasive disease and eventually death despite neoadjuvant chemotherapy and radical cystectomy. We postulate that this may be due to a deleterious underlying somatic gene mutation. Molecular pathologic data obtained on the initial, non-muscle invasive tumor and the final cystectomy specimen, revealed the same TP53 mutation (p.Arg110Pro) in both specimens with a variant allele frequency of 44%. The tumor was tested for 50 common gene mutations in urothelial carcinoma and no other identifiable DNA repair mutations were found, suggesting that this specific TP53 aberration, one that has never been reported in the bladder cancer literature, could be particularly deleterious. Knowing that bladder cancer cell lines that lack TP53 are more resistant to cisplatin and because the tumor lacked any other DNA mutation, this patient may have been a candidate for upfront surgery without neoadjuvant chemotherapy. In addition to histological analysis of the tumor, early molecular and cytogenetic characterization of resected tissue is essential in predicting progression and eventual prognosis of the disease based on identifiable gene mutations. Further comparative prospective studies are required to clarify the importance of molecular heterogeneity and subtyping in bladder cancer.

Background and aims Hepatocellular carcinoma (HCC) is an aggressive malignancy which is often associated with a complex tumor microenvironment attributable to etiology-induced cellular inflammation. γδ T cells are known to detect and react to chronic inflammation, which is linked to cancer development, progression, and metastasis. Our recent genomic study revealed an increased infiltration of several immune cell types, including γδ T cells, in tumor microenvironments of a Thai HCC subtype associated with a good prognosis. Approach and results Here, we quantified the amount of γδ T cells using a γδ T-cell-specific gene signature in 247 Chinese HCC patients. We also validated the γδ T-cell signature in American HCC patients. Additionally, such an association was only found in tumor transcriptomic data, but not in adjacent nontumor transcriptomic data, suggesting a selective enrichment of γδ T cells in the tumor microenvironment. Moreover, the γδ T-cell signature was positively correlated with the expression of natural killer cell receptor genes, such as NKG2D and cytolytic T-cell genes granzymes and perforin, suggesting a stronger T-cell-mediated cytotoxic activity. Furthermore, we found that the γδ T-cell-specific gene expression is positively correlated with the expression of chemokine (C-C motif) ligand 4 (CCL4)/chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor type 1 (CCR1)/C-C chemokine receptor type 5 (CCR5), the receptors for γδ T cells. We validated these results using immunohistochemical analysis of formalin-fixed, paraffin-embedded tumor biopsies from 182 HCC patients. Moreover, we found evidence of CCL4/CCL5-mediated recruitment of γδ T cells both in vitro and in a murine orthotopic Hepa1-6 HCC model. Conclusions We propose that CCL4/CCL5 may interact with their receptor, CCR1/CCR5, which may facilitate the recruitment of γδ T cells from peripheral blood or peritumor regions to the tumor regions. Consequently, an increasing infiltration of γδ T cells in tumors may enhance antitumor immunity and improve patients' prognosis.

Chimeric antigen receptors (CARs) are fusion proteins that contain antigen-recognition domains and T cell signaling domains. Signaling lymphocytic-activation molecule F7 (SLAMF7) is a promising target for CAR T cell therapies of the plasma cell malignancy multiple myeloma (MM) because SLAMF7 is expressed by MM but not normal nonhematopoietic cells. We designed CARs targeting SLAMF7. We transduced human T cells with anti-SLAMF7 CARs containing either CD28 or 4-1BB costimulatory domains. T cells expressing CD28-containing CARs or 4-1BB-containing CARs recognized SLAMF7 in vitro. SLAMF7-specific cytokine release was higher for T cells expressing CARs with CD28 versus 4-1BB domains. In murine solid tumor and disseminated tumor models, anti-tumor activity of T cells was superior with CD28-containing CARs versus 4-1BB-containing CARs. Because of SLAMF7 expression on some normal leukocytes, especially natural killer cells that control certain viral infections, the inclusion of a suicide gene with an anti-SLAMF7 CAR is prudent. We designed a construct with a CD28-containing anti-SLAMF7 CAR and a suicide gene. The suicide gene encoded a dimerization domain fused to a caspase-9 domain. T cells expressing the anti-SLAMF7 CAR plus suicide-gene construct specifically recognized SLAMF7 in vitro and eliminated tumors from mice. T cells expressing this construct were eliminated on demand by administering the dimerizing agent AP1903 (rimiducid).

Background/Aim: Cryptochrome 1 (CRY1), a core circadian gene, modulates circadian rhythm and carcinogenesis. Here, we investigated the role of CRY1 and its correlation with NANOG, a stem cell transcription factor, in cervical cancer. Materials and Methods: Immunohistochemistry with tissue microarray was performed to evaluate CRY1 and NANOG expression in cervical cancer tissues, and their functional roles were assessed in cervical cancer cell lines. Results: CRY1 or NANOG was significantly over-expressed in cervical cancer tissues. Notably, combined over-expression of CRY1 and NANOG was correlated with a significantly poor OS and DFS and showed a stronger predictive value for chemoradiation response than each single protein. Furthermore, siCRY1 induced apoptosis, decreased NANOG expression, suppressed STAT3 signalling, and activated p53 signalling in cervical cancer cell lines. Conclusion: CRY1 and NANOG over-expression serves as a strong predictive biomarker for prognosis and chemoradiation response, and may be a new therapeutic target in patients with cervical cancer.

The application of deep learning for automated segmentation (delineation of boundaries) of histologic primitives (structures) from whole slide images can facilitate the establishment of novel protocols for kidney biopsy assessment. Here, we developed and validated deep learning networks for the segmentation of histologic structures on kidney biopsies and nephrectomies. For development, we examined 125 biopsies for Minimal Change Disease collected across 29 NEPTUNE enrolling centers along with 459 whole slide images stained with Hematoxylin & Eosin (125), Periodic Acid Schiff (125), Silver (102), and Trichrome (107) divided into training, validation and testing sets (ratio 6:1:3). Histologic structures were manually segmented (30048 total annotations) by five nephropathologists. Twenty deep learning models were trained with optimal digital magnification across the structures and stains. Periodic Acid Schiff-stained whole slide images yielded the best concordance between pathologists and deep learning segmentation across all structures (F-scores: 0.93 for glomerular tufts, 0.94 for glomerular tuft plus Bowman's capsule, 0.91 for proximal tubules, 0.93 for distal tubular segments, 0.81 for peritubular capillaries, and 0.85 for arteries and afferent arterioles). Optimal digital magnifications were 5X for glomerular tuft/tuft plus Bowman's capsule, 10X for proximal/distal tubule, arteries and afferent arterioles, and 40X for peritubular capillaries. Silver stained whole slide images yielded the worst deep learning performance. Thus, this largest study to date adapted deep learning for the segmentation of kidney histologic structures across multiple stains and pathology laboratories. All data used for training and testing and a detailed online tutorial will be publicly available.

Public health and health administration programs play a critical role in training future health profession leaders to be strategic and efficacious in this current dynamic environment. As the health care system rapidly transitions from a fee-for-service environment to a value-based delivery system, the role of population health management is a critical concept. A key tool for managing the health of populations requires a health system to discern the community’s problems and resources with the intent of developing, implementing, and evaluating strategies to improve the health of those who live there. The purpose of this work was to (1) engage health educators in a dialogue about the best ways to teach students how to perform a community health needs assessment (CHNA) for the improvement of a population’s health; (2) identify key CHNA data sets; and (3) identify current models and tools, course content, and resources appropriate for undergraduate and graduate health courses when teaching the CHNA process. Authors facilitated group work with participants attending an Educational Session at the annual meeting of the Association of University Programs in Health Administration. This research provides educators with resources to allow them to teach students about CHNAs and how to utilize them when they work for health care organizations.

The emergence of digital pathology — an image-based environment for the acquisition, management and interpretation of pathology information supported by computational techniques for data extraction and analysis — is changing the pathology ecosystem. In particular, by virtue of our new-found ability to generate and curate digital libraries, the field of machine vision can now be effectively applied to histopathological subject matter by individuals who do not have deep expertise in machine vision techniques. Although these novel approaches have already advanced the detection, classification, and prognostication of diseases in the fields of radiology and oncology, renal pathology is just entering the digital era, with the establishment of consortia and digital pathology repositories for the collection, analysis and integration of pathology data with other domains. The development of machine-learning approaches for the extraction of information from image data, allows for tissue interrogation in a way that was not previously possible. The application of these novel tools are placing pathology centre stage in the process of defining new, integrated, biologically and clinically homogeneous disease categories, to identify patients at risk of progression, and shifting current paradigms for the treatment and prevention of kidney diseases., Developments in digital pathology and computational image analysis have the potential to identify new disease mechanisms, improve disease classification and prognostication, and ultimately aid the identification of targeted therapies. In this Review, the authors provide an outline of the digital ecosystem in nephropathology and describe potential applications and challenges associated with the emerging armamentarium of technologies for image analysis., Key points The introduction of digital pathology in clinical research, trials and practice has catalysed the development of novel machine-learning models for tissue interrogation with the potential to improve our ability to discover disease mechanisms, identify comprehensive, patient-specific phenotypes, classify kidney patients into clinically relevant categories, predict disease outcome and, ultimately, identify more targeted therapies.The development of computational image analysis tools for tissue interrogation has brought pathology to the forefront in this process of re-defining kidney diseases.The new nephropathology ecosystem offers several advantages over conventional pathology but also brings some challenges that need to be addressed collectively by all the stake holders, including pathologists, nephrologists, computer scientists, regulatory agencies and patient’s representatives; overcoming these challenges is a pre-requisite for these new machine-learning and computational pathology models to be fully deployed for patient care.The development of novel powerful computational tools for image analysis and data integration in kidney diseases has exposed the need to revise the curriculum for medical professionals to prepare the next generation to fully operate within the new digital pathology ecosystem.Ultimately, our ability to treat kidney diseases (actionable intelligence) will be largely based on the application of artificial (augmenting) intelligence tools and the establishment of synergistic human–machine protocols that integrate digital pathology data with clinical and molecular data for personalized nephrology.

Immune selection drives tumor cells to acquire refractory phenotypes. We previously demonstrated that cytotoxic T lymphocyte (CTL)-mediated immune pressure enriches NANOG(+) tumor cells with stem-like and immune-refractory properties that make them resistant to CTLs. Here, we report that the emergence of refractory phenotypes is highly associated with an aberrant macroautophagic/autophagic state of the NANOG(+) tumor cells and that the autophagic phenotype arises through transcriptional induction of MAP1LC3B/LC3B by NANOG. Furthermore, we found that upregulation of LC3B expression contributes to an increase in EGF secretion. The subsequent hyperactivation of EGFR-AKT signaling rendered NANOG(+) tumor cells resistant to CTL killing. The NANOG-LC3B-p-EGFR axis was preserved across various types of human cancer and correlated negatively with the overall survival of cervical cancer patients. Inhibition of LC3B in immune-refractory tumor models rendered tumors susceptible to adoptive T-cell transfer, as well as PDCD1/PD-1 blockade, and led to successful, long-term control of the disease. Thus, our findings demonstrate a novel link among immune-resistance, stem-like phenotypes, and LC3B-mediated autophagic secretion in immune-refractory tumor cells, and implicate the LC3B-p-EGFR axis as a central molecular target for controlling NANOG(+) immune-refractory cancer. Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A(1); CASP3: caspase 3; CFSE: carboxyfluorescein succinimidyl ester; ChIP: chromatin immunoprecipitation; CI: confidence interval; CIN: cervical intraepithelial neoplasia; CSC: cancer stem cell; CTL: cytotoxic T lymphocyte; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FIGO: International Federation of Gynecology and Obstetrics; GFP: green fluorescent protein; GZMB: granzyme B; HG-CIN: high-grade CIN; IHC: immunohistochemistry; LG-CIN: low-grade CIN; LN: lymph node; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCL1: myeloid cell leukemia sequence 1; MLANA/MART-1: melanoma antigen recognized by T cells 1; MUT: mutant; NANOG: Nanog homeobox; PDCD1/PD-1: programmed cell death 1; PMEL/gp100: premelanosome protein; RTK: receptor tyrosine kinase; TMA: tissue microarray; WT: wild type

OBJECTIVE: Resistance to chemo-radiation therapy is a substantial obstacle that compromises treatment of advanced cervical cancer. The objective of this study was to investigate if a proteomic panel associated with radioresistance could predict survival of patients with locally advanced cervical cancer. METHODS: A total of 181 frozen tissue samples were prospectively obtained from patients with locally advanced cervical cancer before chemoradiation. Expression levels of 22 total and phosphorylated proteins were evaluated using well-based reverse phase protein arrays. Selected proteins were validated with western blotting analysis and immunohistochemistry. Performances of models were internally and externally validated. RESULTS: Unsupervised clustering stratified patients into three major groups with different overall survival (OS, P = 0.001) and progression-free survival (PFS, P = 0.003) based on detection of BCL2, HER2, CD133, CAIX, and ERCC1. Reverse-phase protein array results significantly correlated with western blotting results (R(2) = 0.856). The C-index of model was higher than clinical model in the prediction of OS (C-index: 0.86 and 0.62, respectively) and PFS (C-index: 0.82 and 0.64, respectively). The Kaplan-Meier survival curve showed a dose-dependent prognostic significance of risk score for PFS and OS. Multivariable Cox proportional hazard model confirmed that the risk score was an independent predictor of PFS (HR: 1.6; 95% CI: 1.4–1.9; P < 0.001) and OS (HR: 2.1; 95% CI: 1.7–2.5; P < 0.001). CONCLUSION: A proteomic panel of BCL2, HER2, CD133, CAIX, and ERCC1 independently predicted survival in locally advanced cervical cancer patients. This prediction model can help identify chemoradiation responsive tumors and improve prediction for clinical outcome of cervical cancer patients.

Cancer immunotherapy has emerged as a promising cancer treatment. However, the presence of immune-refractory tumor cells limits its clinical success by blocking amplification of anti-tumor immunity. Previously, we found that immune selection by immunotherapy drives the evolution of tumors toward multi-modal resistant and stem-like phenotypes via transcription induction of AKT co-activator TCL1A by NANOG. Here, we report a crucial role of HSP90A at the crossroads between NANOG-TCL1A axis and multi-aggressive properties of immune-edited tumor cells by identifying HSP90AA1 as a NANOG transcriptional target. Furthermore, we demonstrate that HSP90A potentiates AKT activation through TCL1A-stabilization, thereby contributing to the multi-aggressive properties in NANOGhigh tumor cells. Importantly, HSP90 inhibition sensitized immune-refractory tumor to adoptive T cell transfer as well as PD-1 blockade, and re-invigorated the immune cycle of tumor-reactive T cells. Our findings implicate that the HSP90A-TCL1A-AKT pathway ignited by NANOG is a central molecular axis and a potential target for immune-refractory tumor., Nanog can confer resistance to cancer immunotherapy by promoting AKT activity. Here, the authors demonstrate that HSP90A is a Nanog target that stabilizes the AKT coactivator TCL1, thereby activating AKT, and that HSP90A inhibition can enhance the anti-tumor efficacy of adoptive T cell transfer and checkpoint blockade.

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy., Metastatic cells can mimic many of the phenotypic behaviors of embryonic cells. Here, the authors generate a melanoblast-specific transcriptome using a genetically engineered mouse model and identify KDELR3 as a pro-metastasis gene in melanoma.

Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin–stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm2, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm2, ORT3vsT1: 3.42, 95% confidence interval (CI), 1.87–6.28; acini count/TDLUT3vsT1, OR: 2.40, 95% CI, 1.39–4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.

Objective: To determine if a weight gain prevention intervention is acceptable to young women with a normal Body Mass Index and a moderately increased or high risk of breast cancer. Design: Qualitative semi-structured interview study involving 14 women aged 26–35 years who were registered with a Family History Clinic in Manchester, UK, due to family history of breast cancer. Participants’ views were analysed thematically. Results: Four themes were produced: (1) perceptions of a healthy lifestyle: women’s perceptions included health-related behaviours and subjective wellbeing; (2) construing a healthy weight: women rely on appearance, feelings and others opinions to construe weight instead of quantitative indicators; (3) configuring a useful programme: the idea of a programme that is remotely accessible; provides a point of contact; and promotes general wellbeing was appealing. Women believed information explaining the link between lifestyle and breast cancer would facilitate behaviour change; (4) the importance of will(power): women recognised that commitment to a programme is affected by time, money and readiness to change. Conclusion: A weight gain prevention intervention that focuses on wellbeing and behaviour change appears acceptable to many healthy weight women. Future research should examine whether women’s expressed acceptability translates into actual acceptability of such a programme.

PURPOSE Genetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)–associated epithelial cancers. METHODS This phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin. RESULTS Twelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient’s resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell–mediated antitumor activity. Another patient’s resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation. CONCLUSION Engineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.

PURPOSE: Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) have been reported to play an imported role in human cancers, but their role in cervical cancer has not yet been clarified. In this study, we evaluated the functional role of FOXO1 in cervical cancer cells and investigated the expression and clinical significance of FOXO1 and PAX3 in cervical lesions. METHODS: In vitro assessment of cell function by cell viability, migration, and invasion assays were performed on FOXO1-knockdown cervical cancer cells. Immunohistochemical (IHC) staining analyses of FOXO1 and PAX3 were performed with a tissue microarray (TMA). The clinical significance was evaluated by comparing the data with various clinicopathologic characteristics, including survival of patients with cervical cancer. RESULTS: In vitro results revealed that knockdown of FOXO1 is associated with decreased cell viability (p < 0.001), migration (p < 0.001), and invasion (p < 0.05), supporting the oncogenic role of FOXO1 in cervical cancer. FOXO1 and PAX3 expression was significantly higher in CIN (both p < 0.001) and cancer tissue (both p < 0.001) than in normal tissue. Multivariate analysis indicated that FOXO1 expression (hazard ratio 4.01 [95% CI 1.22–13.10], p = 0.021) and an advanced FIGO stage (hazard ratio 3.89 [95% CI 1.35–11.19], p = 0.012) were independent prognostic factors for overall survival. CONCLUSIONS: This study reveals increased FOXO1 and PAX3 expression in cervical cancers and indicates an oncogenic role of FOXO1 in cervical cancer cells that correlates with poor patient survival.

Proteus syndrome is a mosaic, progressive overgrowth disorder caused by a somatic activating variant c.49G > A p.(E17K) in AKT1. The presentation in affected individuals is variable, with a diversity of tissues demonstrating abnormalities. Common manifestations include skin and bony overgrowth, vascular malformations (VMs), cysts and benign tumors. We used two methods to create mouse models that had endogenously-regulated mosaic expression of the Proteus syndrome variant. Variant allele fractions (VAFs) ranged from 0% to 50% across numerous tissues in 44 Proteus syndrome mice. Mice were phenotypically heterogeneous with lesions rarely observed before 12 months of age. VMs were the most frequent finding with a total of 69 found in 29 of 44 Proteus syndrome mice. Twenty-eight cysts and ectasia, frequently biliary, were seen in 22 of 44 Proteus syndrome mice. Varying levels of mammary hyperplasia were seen in 10 of 16 female Proteus syndrome mice with other localized regions of hyperplasia and stromal expansion noted in several additional animals. Interestingly, 27 of 31 Proteus syndrome animals had non-zero blood VAF that is in contrast to the human disorder where it is rarely seen in peripheral blood. Identification of variant-positive cells by green fluorescent protein (GFP) staining in chimeric Proteus syndrome mice showed that in some lesions, hyperplastic cells were predominantly GFP/Akt1E17K-positive and showed increased pAKT signal compared to GFP-negative cells. However, hyperplastic mammary epithelium was a mixture of GFP/Akt1E17K-positive and negative cells with some GFP/Akt1E17K-negative cells also having increased pAKT signal suggesting that the variant-positive cells can induce lesion formation in a non-cell autonomous manner.

Aim: We have shown that IL-4 fused to Pseudomonas exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. Materials & Methods: Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. Results: The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. Conclusion: The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.

The current classification system for focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) does not fully capture the complex structural changes in kidney biopsies, nor the clinical and molecular heterogeneity of these diseases. The Nephrotic Syndrome Study Network (NEPTUNE) Digital Pathology Scoring System (NDPSS) was applied to 221 NEPTUNE FSGS/MCD digital kidney biopsies for glomerular scoring using 37 descriptors. The descriptor-based glomerular profiles were used to cluster patients with similar morphologic characteristics. Glomerular descriptors and patient clusters were assessed for association with time to proteinuria remission and disease progression by using adjusted Cox models, and eGFR measures over time by using linear mixed models. Messenger RNA from glomerular tissue was used to assess differentially expressed genes (DEG) between clusters and identify genes associated with individual descriptors driving cluster membership. Three clusters were identified: X (N=56), Y (N=68), and Z (N=97). Clusters Y and Z had higher probabilities of proteinuria remission (HR [95% CI]= 1.95 [0.99, 3.85] and 3.29 [1.52, 7.13], respectively), lower hazards of disease progression 0.22 [0.08, 0.57] and 0.11 [0.03, 0.45], respectively), and greater loss of eGFR over time compared with X. Cluster X had 1920 DEGs compared to Y+Z, which reflected activation of pathways of immune response and inflammation. Six individual descriptors driving the clusters individually correlated with clinical outcomes and gene expression. The NDPSS allows for characterization of FSGS/MCD patients into clinically and biologically relevant categories and uncovers histologic parameters associated with clinical outcomes and molecular signatures not included in current classification systems.TRANSLATIONAL STATEMENTFSGS and MCD are heterogeneous diseases that manifest with a variety of structural changes often not captured by conventional classification systems. This study shows that a detailed morphologic analysis and quantification of these changes allows for better representation of the structural abnormalities within each patient and for grouping patients with similar morphologic profiles into categories that are clinically and biologically relevant.

Purpose: The Cancer Immune Monitoring and Analysis Centers – Cancer Immunologic Data Commons (CIMAC-CIDC) network supported by the NCI Cancer Moonshot initiative was established to provide correlative analyses for clinical trials in cancer immunotherapy, using state-of-the-art technology. Fundamental to this initiative is implementation of multiplex IHC assays to define the composition and distribution of immune infiltrates within tumors in the context of their potential role as biomarkers. A critical unanswered question involves the relative fidelity of such assays to reliably quantify tumor-associated immune cells across different platforms. Experimental Design: Three CIMAC sites compared across their laboratories: (i) image analysis algorithms, (ii) image acquisition platforms, (iii) multiplex staining protocols. Two distinct high-dimensional approaches were employed: multiplexed IHC consecutive staining on single slide (MICSSS) and multiplexed immunofluorescence (mIF). To eliminate variables potentially impacting assay performance, we completed a multistep harmonization process, first comparing assay performance using independent protocols followed by the integration of laboratory-specific protocols and finally, validating this harmonized approach in an independent set of tissues. Results: Data generated at the final validation step showed an intersite Spearman correlation coefficient (r) of ≥0.85 for each marker within and across tissue types, with an overall low average coefficient of variation ≤0.1. Conclusions: Our results support interchangeability of protocols and platforms to deliver robust, and comparable data using similar tissue specimens and confirm that CIMAC-CIDC analyses may therefore be used with confidence for statistical associations with clinical outcomes largely independent of site, antibody selection, protocol, and platform across different sites.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

Simple Summary Functionality of the cellular DNA damage response (DDR) network affects risk for developing cancer, and the DDR is also a target of cancer therapies. Thus, it is important that we have reliable laboratory methods for determining the activity of this network. We describe the development and analytical validation of a targeted mass spectrometry-based 51-plex assay (DDR-2) for measuring proteins and post-translational modifications related to the DDR. The findings demonstrate identification of potential novel pharmacodynamic biomarkers. Abstract The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Hormone receptor expression patterns often correlate with infiltration of specific lymphocytes in tumors. Specifically, the presence of specific tumor-infiltrating lymphocytes (TILs) with particular hormone receptor expression is reportedly associated with breast cancer, however, this has not been revealed in epithelial ovarian cancer (EOC). Therefore, we investigated the association between hormone receptor expression and TILs in EOC. Here we found that ERα, AR, and GR expression increased in EOC, while PR was significantly reduced and ERβ expression showed a reduced trend compared to normal epithelium. Cluster analysis indicated poor disease-free survival (DFS) in AR+/GR+/PR+ subgroup (triple dominant group), while the Cox proportional-hazards model highlighted the triple dominant group as an independent prognostic factor for DFS. In addition, significant upregulation of FoxP3+ TILs, PD-1, and PD-L1 was observed in the triple dominant group compared to other groups. NanoString analyses further suggested that tumor necrosis factor (TNF) and/or NF-κB signaling pathways were activated with significant upregulation of RELA, MAP3K5, TNFAIP3, BCL2L1, RIPK1, TRAF2, PARP1, and AKT1 in the triple dominant EOC group. The triple dominant subgroup correlates with poor prognosis in EOC. Moreover, the TNF and/or NF-κB signaling pathways may be responsible for hormone-mediated inhibition of the immune microenvironment.

All-digital infrared spectroscopic imaging of tumors and their microenvironments predicts colon cancer patient survival., The structure and organization of a tumor and its microenvironment are often associated with cancer outcomes due to spatially varying molecular composition and signaling. A persistent challenge is to use this physical and chemical spatial organization to understand cancer progression. Here, we present a high-definition infrared imaging–based organizational measurement framework (INFORM) that leverages intrinsic chemical contrast of tissue to label unique components of the tumor and its microenvironment. Using objective and automated computational methods, further, we determine organization characteristics important for prediction. We show that the tumor spatial organization assessed with this framework is predictive of overall survival in colon cancer that adds to capability from clinical variables such as stage and grade, approximately doubling the risk of death in high-risk individuals. Our results open an all-digital avenue for measuring and studying the association between tumor spatial organization and disease progression.

Purpose: Immunoprofiling to identify biomarkers and integration with clinical trial outcomes are critical to improving immunotherapy approaches for patients with cancer. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assay performance further limits comparison and interpretation of data across studies and laboratories. Experimental Design: To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer MoonshotSM Initiative of the National Cancer Institute (NCI) and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the NIH. Results: The CIMAC-CIDC Network is composed of four academic centers with multidisciplinary expertise in cancer immunotherapy that perform validated and harmonized assays for immunoprofiling and conduct correlative analyses. A data coordinating center (CIDC) provides the computational expertise and informatics platforms for the storage, integration, and analysis of biomarker and clinical data. Conclusions: This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure, validation and harmonization of core immunoprofiling assays, platforms for data ingestion and integration, and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole-exome sequencing, RNA sequencing, cytometry by time of flight, and IHC/immunofluorescence.

BACKGROUND: Aberrant activation of the WNT/β-catenin and STAT3 signaling pathways plays a critical role in cancer progression. However, direct targeting of these pathways as an anti-cancer therapeutic approach needs to be reconsidered due to its serious side effects. Here, we demonstrate that overexpression of WNT induces STAT3 activation in a galectin-3-dependent manner. METHODS: We investigated how galectin-3 mediates the crosstalk between WNT/β-catenin and STAT3 signaling and whether inhibition of galectin-3 can reduce gastric cancer. The molecular mechanisms were analyzed by biochemical assays using cultured gastric cancer cells, patient tissues, and genetically engineered mice. Moreover, we confirm of therapeutic effects of GB1107, a cell-penetrating galectin-3 specific inhibitor, using orthotopic gastric cancer-bearing mice RESULTS: Increased levels of galectin-3 and STAT3 phosphorylation were detected in the stomach tissues of WNT1-overexpressing mouse models. Also, high expression levels and co-localization of β-catenin, pSTAT3, and galectin-3 in patients with advanced gastric cancer were correlated with a poorer prognosis. Galectin-3 depletion significantly decreased STAT3 Tyr705 phosphorylation, which regulates its nuclear localization and transcriptional activation. A peptide of galectin-3 (Y45-Q48) directly bound to the STAT3 SH2 domain and enhanced its phosphorylation. GB1107, a specific membrane-penetrating inhibitor of galectin-3, significantly reduced the activation of both STAT3 and β-catenin and inhibited tumor growth in orthotopic gastric cancer-bearing mice. CONCLUSIONS: We propose that galectin-3 mediates the crosstalk between the WNT and STAT3 signaling pathways. Therefore GB1107, a galectin-3-specific inhibitor, maybe a potent agent with anti-gastric cancer activity. Further studies are needed for its clinical application in gastric cancer therapy.

BACKGROUND The aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention. METHODS Individuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3–12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing. RESULTS Twenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses. CONCLUSION To our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent. TRIAL REGISTRATION NCT02581137 FUNDING NIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870

Background Nutrition and mealtime interventions can improve nutritional intake amongst hospital inpatients; however, patient-reported experience is rarely considered in their development and evaluation. The present study aimed to measure patient-reported food and mealtime experience to evaluate and inform continuous quality improvement of hospital nutrition care. Methods A cross-sectional survey with inpatients in seven acute care and rehabilitation wards was conducted. A 27-item validated questionnaire measured five domains of patient experience: food choices, organisational barriers, feeling hungry, physical barriers to eating and food quality. Responses were summarised descriptively and compared between settings (acute versus rehabilitation), patient demographics (age, gender) and time in hospital. Results Responses from 143 participants (mean age 67 years, 57% male, 28% rehabilitation, median 6 days into hospitalisation) showed that 10% or fewer respondents reported difficulties with food choices, feeling hungry or food quality. The most common difficulties were opening packets (36%), insufficient menu information provided (29%), being interrupted by staff when eating (28%), being disturbed when eating (27%), being in an uncomfortable position when eating (24%) and difficulty reaching food (21%). There were no significant differences in domain patterns by sex, age group or time in hospital. Organisational barriers were reported less frequently amongst rehabilitation participants compared to acute care (P = 0.01). Conclusions This survey highlights areas of positive patient-reported experience with nutrition care and suggests that local improvement efforts should focus on physical assistance needs and organisational barriers, especially in acute care wards. The questionnaire may be useful for informing and evaluating systematic nutrition care improvements.

Background The tumor microenvironment (TME) is a critical player in tumor progression, metastasis and therapy outcomes. Tumor-associated macrophages (TAMs) are a well-recognized core element of the TME and generally characterized as M2-like macrophages. TAMs are believed to contribute to tumor progression, but the mechanism behind this remains unclear. We aimed to investigate the clinical, angiogenic, and lymphangiogenic significance of TAMs in non-small cell lung cancer (NSCLC). Methods Utilizing combined immunohistochemistry and digital image analysis, we assessed CD68, CD163, VEGF-A, and VEGF-C expression in 349 patients with NSCLC. Subsequently, the potential association between M2 TAMs and angiogenic VEGF-A and/or lymphangiogenic VEGF-C was evaluated for its prognostic value. Furthermore, the effects of M2 TAMs on angiogenesis and lymphangiogenesis were explored via an in vitro co-culture system. Results CD68 and CD163 expression were found to directly correlate with VEGF-A and/or VEGF-C expression (all p p = 0.023). Dual expression of M2 ratiohigh and VEGF-Chigh (M2 ratiohighVEGF-Chigh) was correlated with worse overall survival (p = 0.033). Multivariate analysis revealed that M2 ratiohigh [HR (95% CI) = 1.53 (1.01–2.33), p = 0.046] and combined M2 ratiohighVEGF-Chigh expression [HR (95% CI) = 2.01 (1.28–3.16), p = 0.003] were independent predictors of poor overall survival. Notably, we confirmed that M2 macrophages significantly enhanced the protein and mRNA expression of both VEGF-A and VEGF-C, while M1 macrophages induced only mRNA expression of VEGF-A in A549 cells. Conclusions This study suggests that TAMs are significantly associated with angiogenesis and lymphangiogenesis, contributing to the progression of NSCLC. Furthermore, elevated M2 ratio, similar to combined high M2 ratio and high VEGF-C expression, is a strong indicator of poor prognosis in patients with NSCLC, providing insight for future TAM-based immunotherapy strategies.

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS-CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibitingACE2expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission.

Terminal duct lobular units (TDLUs) are the predominant anatomical structures where breast cancers originate. Having lesser degrees of age-related TDLU involution, measured as higher TDLUs counts or more epithelial TDLU substructures (acini), is related to increased breast cancer risk among women with benign breast disease (BBD). We evaluated whether a recently developed polygenic risk score (PRS) based on 313-common variants for breast cancer prediction is related to TDLU involution in the background, normal breast tissue, as this could provide mechanistic clues on the genetic predisposition to breast cancer. Among 1398 women without breast cancer, higher values of the PRS were significantly associated with higher TDLU counts (P = 0.004), but not with acini counts (P = 0.808), in histologically normal tissue samples from donors and diagnostic BBD biopsies. Mediation analysis indicated that TDLU counts may explain a modest proportion (≤10%) of the association of the 313-variant PRS with breast cancer risk. These findings suggest that TDLU involution might be an intermediate step in the association between common genetic variation and breast cancer risk.

Lipid peroxidation is a common feature of liver diseases, especially non-alcoholic fatty liver disease (NAFLD). There are limited validated tools to study intra-hepatic lipid peroxidation, especially for small specimen. We developed a semi-quantitative, fully automated immunohistochemistry assay for the detection of 4-hydroxynoneal (4-HNE) protein adducts, a marker of lipid peroxidation, for adaptation to clinical diagnostics and research. We used Hep G2 cells treated with 4-HNE to validate specificity, sensitivity, and dynamic range of the antibody. Staining and semi-quantitative automated readout were confirmed in human needle-biopsy liver samples from subjects with NAFLD and normal liver histology. The ability to detect changes in lipid peroxidation was tested in paired liver biopsies from NAFLD subjects, obtained before and after 4 weeks of treatment with the antioxidant vitamin E (ClinicalTrials.gov NCT01792115, n=21). The cellular calibrator was linear and NAFLD patients had significantly higher levels of 4-HNE adducts compared to controls ( p=0.02). Vitamin E treatment significantly decreased 4-HNE ( p=0.0002). Our findings demonstrate that 4-HNE quantification by immunohistochemistry and automated image analysis is feasible and able to detect changes in hepatic lipid peroxidation in clinical trials. This method can be applied to archival and fresh samples and should be considered for use in assessing NAFLD histology.

Objective: Hairy and enhancer of split-1 (HES-1), which is a downstream target of the Notch signaling pathway, has been linked to KRAS mutations. HES-1 has been proposed as harboring oncogenic activity in colorectal cancer but has not been investigated in adenocarcinoma of the small intestine, where the drivers of oncogenesis are not as well understood. Materials and Methods: To investigate the clinicopathologic and prognostic implications of HES-1, HES-1 immunohistochemical expression was analyzed in digital images along with clinicopathological variables, including survival and KRAS genotype, in 185 small intestinal adenocarcinomas. Results: The loss of HES-1 expression (HES-1Loss) was observed in 38.4% (71/185) of the patients, and was associated with higher pT category (P = 0.018), pancreatic invasion (P = 0.005), high grade (P =0.043), and non-tubular histology (P = 0.004). Specifically, in tumors with mutant KRAS (KRASMT), HES-1Loss was related to proximal location (P = 0.024), high T and N categories (P = 0.005 and P = 0.047, respectively), and pancreatic invasion (P = 0.004). Patients with HES-1Loss showed worse overall survival compared to those with intact HES-1 (HES-1Intact) (P = 0.013). Patients with HES-1Loss/KRASMT (median, 17.3 months) had significantly worse outcomes than those with HES-1Intact/KRASWT (39.9 months), HES-1Intact/KRASMT (47.6 month), and HES-1Loss/KRASWT (36.2 months) (P = 0.010). By multivariate analysis, HES-1Loss (hazard ratio = 1.55, 95% confidence interval (CI), 1.07 – 2.26; P = 0.022) remained an independent prognostic factor. Conclusion: HES-1 expression can be used as a potential prognostic marker and may aid in the management of patients with small intestinal adenocarcinomas.

Background/objectives Older people living in residential aged care facilities (RACFs) experience acute deterioration requiring assessment and decision making. We evaluated the impact of a large-scale regional Aged Care Emergency (ACE) program in reducing hospital admissions and emergency department (ED) transfers. Design A stepped wedge nonrandomized cluster trial with 11 steps, implemented from May 2013 to August 2016. Setting A large regional and rural area of northern and western New South Wales, Australia. Participants Nine hospital EDs and 81 RACFs participated in the evaluation. Intervention The ACE program is an integrated nurse-led intervention underpinned by a community of practice designed to improve the capability of RACFs managing acutely unwell residents. It includes telephone support, evidence-based algorithms, defining goals of care for ED transfer, case management in the ED, and an education program. Measurements ED transfers and subsequent hospital admissions were collected from administrative data including 13 months baseline and 9 months follow-up. Results A total of 18,837 eligible ED visits were analyzed. After accounting for clustering by RACFs and adjusting for time of the year as well as RACF characteristics, a statistically significant reduction in hospital admissions (adjusted incident rate ratio = .79; 95% confidence interval [CI] = .68-.92); P = .0025) was seen (i.e., residents were 21% less likely to be admitted to the hospital). This was also observed in ED visit rates (adjusted incidence rate ratio = .80; 95% CI = .69-.92; P = .0023) (i.e., residents were 20% less likely to be transferred to the ED). Seven-day ED re-presentation fell from 5.7% to 4.9%, and 30-day hospital readmissions fell from 12% to 10%. Conclusion The stepped wedge design allowed rigorous evaluation of a real-world large-scale intervention. These results confirm that the ACE program can be scaled up to a large geographic area and can reduce ED visits and hospitalization of older people with complex healthcare needs living in RACFs.

BACKGROUND: Epstein-Barr virus (EBV) positivity is associated with better gastric cancer prognosis and is found in a relatively fixed 9% of tumors worldwide. AIMS: We aimed to examine the EBV status of gastric adenocarcinomas in a very high-incidence population and to compare prevalence between cardia and non-cardia anatomic subsites. METHODS: We evaluated 1035 adult gastric adenocarcinoma cases presenting during 1997-2005 to the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, China. EBV-encoded RNA was detected in alcohol-fixed paraffin-embedded tumor specimens by in situ hybridization. Associations were assessed in case-case comparisons using the chi-squared test for categorical variables and the Mann-Whitney U Test for continuous variables, with p-values < 0.05 considered statistically significant. Adjusted odds ratios were calculated using logistic regression, and mortality hazard ratios (HRs) were estimated by Cox proportional hazards regression. RESULTS: Sixty-four percent of the evaluated cancers were found in the cardia. Cardia tumor localization was associated with male sex, advanced primary tumor stage, better differentiated histology, and intestinal-type Lauren classification. Four percent of the non-cardia and only 0.9% of cardia cancers were EBV-positive. EBV-positivity was associated with better overall survival (adjusted HR 0.30, 95% CI 0.14-0.63). CONCLUSIONS: Our study highlights unusually low EBV prevalence in gastric adenocarcinoma among a high-incidence population, particularly for cardia cancers. These findings suggest a unique risk factor profile for the high incidence of gastric cancer in this population.

3125 Background: The Cancer Immune Monitoring and Analysis Centers Cancer Immunology Data Commons (CIMAC-CIDC) network is a NCI Cancer Moonshots initiative to provide state-of-the-art technology and expertise for immunotherapy clinical trials. Multiplex tissue immunostaining is an integral assay provided that examines density and spatial distribution of immune cells and markers in tissues, for their prognostic or predictive value. Two approaches were evaluated for sensitivity, specificity, and reproducibility and subsequently harmonized: chromogenic-based Multiplex Immunohistochemical Consecutive Staining on Single Slide (MICSSS) and Multiplex Immunofluorescence (mIF) based tyramide signal amplification system. Methods: Harmonization was performed across CIMACs (Mount Sinai, Dana Farber Cancer Institute, MD Anderson Cancer Center) in multiple steps to prove that comparable data can be generated independent of site and platform. Goals: 1) harmonize image analysis platforms alone using tissues pre-stained with single chromogenic IHC for CD3 (membrane), Ki67 (nuclear), and CD68 (cytoplasmic), 2) compare image acquisition platforms, 3) streamline Antibody (Ab) clones and assess PD-L1 detection in relation to CLIA- assays, 4) harmonize staining protocols, image acquisition, and analysis platforms on 2 test head and neck tumor samples using MICSSS and mIF, 5) validate harmonization results with a tissue microarray on 27 tissues representing multiple tumors. For last steps, each CIMAC used their platforms for PD-L1, PD-1, CD3, CD8, and pan-cytokeratin (PanCK) staining on one of three consecutive slides from serial sections and compared densities of each marker. Results: Variables as PD-1 Ab clone, positive control reference tissues, sigma value for nuclear segmentation, and use of machine-learning based cell classifier were found to be key to produce accurate, reliable, comparable data. After visual quality control assessment and comparisons of each Region Of Interest (ROI), an overall inter-site Spearman correlation coefficient of ≥0.85 was achieved per marker within each tissue and across tissue types (expect pan-Cytokeratin, ≥0.7), with average coefficient of variation ≤0.1. Conclusions: These results show for the first time that two platforms can deliver harmonized data, despite differences in protocols, platforms, reagents, and analysis tools. Data resulting from retrospective and prospective CIMAC-CIDC analyses may be used with confidence for statistical associations with clinical parameters and outcome.

Marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) belong to a subgroup of indolent B-cell lymphomas most commonly reported in the canine spleen. The goal of this study was to characterize the immunophenotype of splenic MZL and MCL in comparison to their human counterparts. Ten MCLs and 28 MZLs were selected based on morphology. A tissue microarray was generated, and expression of CD3, CD5, CD10, CD45, CD20, CD79a, Pax-5, Bcl-2, Bcl-6, cyclin D1, cyclin D3, MCL-1, MUM-1, and Sox-11 was evaluated. Neoplastic cells in all MCLs and MZLs were positive for CD5, CD20, CD45, CD79a, and BCL2 and negative for CD3, CD10, Bcl-6, cyclin D1, and cyclin D3. Positive labeling for Pax-5 was detected in 8 of 10 MCLs and 26 of 28 MZLs. Positive labeling for MUM-1 was detected in 3 of 10 MCLs, and 27 of 28 MZLs were positive for MUM-1. No MCLs but 8 of 24 MZLs were positive for MCL-1. Canine splenic MZL and MCL have a similar immunophenotype as their human counterparts. However, human splenic MCL overexpresses cyclin D1 due to a translocation. A similar genetic alteration has not been reported in dogs. In addition, in contrast to human MZL, canine splenic MZL generally expresses CD5. Following identification of B vs T cells with CD20 and CD3, a panel composed of BCL-2, Bcl-6, MUM-1, and MCL-1 combined with the histomorphological pattern can be used to accurately diagnose MZL and MCL in dogs. Expression of Bcl-2 and lack of MCL-1 expression in MCL may suggest a therapeutic benefit of BCL-2 inhibitors in canine MCL.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen endemic to dromedary camels known to cause a respiratory illness in humans that is fatal in 35% of cases As such, inactivated tissues provide a safer format when testing for this virus Neutral buffered formalin (NBF), pathology?s gold standard for tissue fixation, preserves histomorphology and inactivates many pathogens However, NBF causes the crosslinking and fragmentation of proteins and nucleic acids in tissues This results in a rapid decline in biomolecular quality after just 24 hours and damage increases thereafter NBF is also a volatile organic compound and carcinogen Two novel fixatives, phosphate-buffered 70% ethanol (BE70) and BE70 with added guanidium salts (BE70G), are alternative preservatives that cause less damage to biomolecules, even in tissues immersed for longer than 3 months These ethanol (EtOH)-based fixatives have equivalent histomorphology and inactivation profiles as well as better human safety profiles We hypothesized that existing anti-MERS-CoV immunohistochemistry (IHC) and in situ hybridization (ISH) protocols would work on tissues preserved with these EtOH-based fixatives Roughly equivalent tracheal and nasal turbinate samples from eight alpacas experimentally infected with MERS-CoV were fixed in 10% NBF, BE70, and BE70G, processed into paraffin and sectioned for hematoxylin and eosin staining or immunolabeling Anti-MERS-CoV nucleocapsid IHC using polymer-based detection and MERS-CoV specific ISH using RNAscope? 2 5 HD brown detection were conducted on all samples Because many EtOH-based fixatives do not require antigen retrieval (AR), ISH and IHC were run with and without AR on the EtOH-based fixed tissues Although MERS-CoV IHC and ISH appropriately labeled target molecules in all samples, signal strength varied across the fixatives The strongest labeling was seen in NBF, followed by BE70G, and finally BE70 This result was not unexpected as the protocols were optimized for use with NBF tissues Interestingly, both BE70 and BE70G required AR to successfully visualize the targets by both IHC and ISH, although BE70G did have weak ISH signal without AR Histomorphology for all three fixatives without AR was very similar However, AR blurred cell borders and reduced overall cellular definition in the EtOH-based fixed samples, particularly BE70G Taken together, these results support the use of these novel EtOH-based fixatives as alternatives to NBF, particularly when biomolecule preservation for downstream molecular biology assays is critical Finally, BE70 and BE70G increase human safety by inactivating pathogens with less harmful chemicals as well as provide a more flexible fixation time frame This is particularly useful for high containment work as it would allow safe transfer of inactivated tissues to lower biosafety level labs while better preserving the samples for downstream analysis Support or Funding Information This work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University and the USDA APHIS NBAF Scientist Training Program Histomorphology, Immunohistochemistry (IHC), and In Situ Hybridization (ISH) of NBF-, BE70-, and BE70G-Fixed Tissues Column 1 is hematoxylin and eosin staining, Column 2 is anti-MERS-CoV nucleocapsid IHC, and Column 3 is MERS-CoV-specific ISH in fixed, paraffin-embedded nasal turbinates All immunolabeled tissues were counterstained with hematoxylin Row 1 is NBF-fixed tissue Row 2 is BE70-fixed tissue Row 3 is BE70G-fixed tissue The bar for all main images is 100 microns

Ovarian clear cell adenocarcinoma (Ov-CCA) has a higher prevalence in the Japanese ancestry than other populations. The ancestral disparities in Ov-CCA prevalence suggests the presence of Ov-CCA-specific genetic alterations and may provide an opportunity to identify the novel genes associated with Ov-CCA tumorigenesis. Using 94 previously reported genes as the phenotypic trait, we conducted multistep expression quantitative trait loci (eQTL) analysis with the HapMap3 project datasets. Four single-nucleotide polymorphisms (SNPs) (rs4873815, rs12976454, rs11136002, and rs13259097) that had different allele frequencies in the Japanese ancestry and seven genes associated in cis (APBA3, C8orf58, KIAA1967, NAPRT1, RHOBTB2, TNFRSF10B, and ZNF707) were identified. In silico functional annotation analysis and in vitro promoter assay validated the regulatory effect of rs4873815-TT on ZNF707 and rs11136002-TT on TNFRSF10B. Furthermore, ZNF707 was highly expressed in Ov-CCA and had a negative prognostic value in disease recurrence in our sample cohort. This prognostic power was consistently observed in The Cancer Genome Atlas (TCGA) clear cell renal cell carcinoma dataset, suggesting that ZNF707 may have prognostic value in clear cell histology regardless of tissue origin. In conclusion, rs4873815-TT/ZNF707 may have clinical significance in the prognosis and tumorigenesis of Ov-CCA, which may be more relevant to clear cell histology. Besides, this study may underpin the evidence that cis-eQTL analysis based on ancestral disparities can facilitate the discovery of causal genetic alterations in complex diseases, such as cancer.

Immuno-therapeutics aim to activate the body's own immune system against cancer and are one of the most promising cancer treatment strategies, but currently limited by a variable response rate. Biomarkers may help to distinguish those patients most likely to respond to therapy; they may also help guide clinical decision making for combination therapies, dosing schedules, and determining progression versus relapse. However, there is a need to confirm such biomarkers in preferably prospective clinical trials before they can be used in practice. Accordingly, it is essential that clinical trials for immuno-therapeutics incorporate biomarkers. Here, focusing on the specific setting of immune therapies, we discuss both the scientific and logistical hurdles to identifying potential biomarkers and testing them in clinical trials.

Limited epidemiological evidence suggests that the etiology of hormone receptor positive (HR+) breast cancer may differ by levels of histologic grade and proliferation. We pooled risk factor and pathology data on 5,905 HR+ breast cancer cases and 26,281 controls from 11 epidemiological studies. Proliferation was determined by centralized automated measures of KI67 in tissue microarrays. Odds ratios (OR), 95% confidence intervals (CI) and p-values for case-case and case-control comparisons for risk factors in relation to levels of grade and quartiles (Q1-Q4) of KI67 were estimated using polytomous logistic regression models. Case-case comparisons showed associations between nulliparity and high KI67 [OR (95% CI) for Q4 vs. Q1=1.54 (1.22, 1.95)]; obesity and high grade [grade 3 vs. 1=1.68 (1.31, 2.16)] and current use of combined hormone therapy (HT) and low grade [grade 3 vs. 1=0.27 (0.16, 0.44)] tumors. In case-control comparisons, nulliparity was associated with elevated risk of tumors with high but not low levels of proliferation [1.43 (1.14, 1.81) for KI67 Q4 vs. 0.83 (0.60, 1.14) for KI67 Q1]; obesity among women ≥50 years with high but not low grade tumors [1.55 (1.17, 2.06) for grade 3 vs. 0.88 (0.66, 1.16) for grade 1] and HT with low but not high grade tumors [3.07 (2.22, 4.23) for grade 1 vs. 0.85 (0.55, 1.30) for grade 3]. Menarcheal age and family history were similarly associated with HR+ tumors of different grade or KI67 levels. These findings provide insights into the etiologic heterogeneity of HR+ tumors.

Context Testing reproducibility is critical for the development of methodologies for morphologic assessment. Our previous study using the descriptor-based Nephrotic Syndrome Study Network Digital Pathology Scoring System (NDPSS) on glomerular images revealed variable reproducibility. Objective To test reproducibility and feasibility of alternative scoring strategies for digital morphologic assessment of glomeruli and explore use of alternative agreement statistics. Design The original NDPSS was modified (NDPSS1 and NDPSS2) to evaluate (1) independent scoring of each individual biopsy level, (2) use of continuous measures, (3) groupings of individual descriptors into classes and subclasses prior to scoring, and (4) indication of pathologists' confidence/uncertainty for any given score. Three and 5 pathologists scored 157 and 79 glomeruli using the NDPSS1 and NDPSS2, respectively. Agreement was tested using conventional (Cohen κ) and alternative (Gwet agreement coefficient 1 [AC1]) agreement statistics and compared with previously published data (original NDPSS). Results Overall, pathologists' uncertainty was low, favoring application of the Gwet AC1. Greater agreement was achieved using the Gwet AC1 compared with the Cohen κ across all scoring methodologies. Mean (standard deviation) differences in agreement estimates using the NDPSS1 and NDPSS2 compared with the single-level original NDPSS were −0.09 (0.17) and −0.17 (0.17), respectively. Using the Gwet AC1, 79% of the original NDPSS descriptors had good or excellent agreement. Pathologist feedback indicated the NDPSS1 and NDPSS2 were time-consuming. Conclusions The NDPSS1 and NDPSS2 increased pathologists' scoring burden without improving reproducibility. Use of alternative agreement statistics was strongly supported. We suggest using the original NDPSS on whole slide images for glomerular morphology assessment and for guiding future automated technologies.

Objective:To determine the prevalence and features of fatty liver disease in spinal and bulbar muscular atrophy (SBMA).Methods:Two groups of participants with SBMA were evaluated. In the first group, 22 participants with SBMA underwent laboratory analysis and liver imaging. In the second group, 14 participants with SBMA were compared to 13 female carriers and 23 controls. Liver biopsies were done in 4 participants with SBMA.Results:Evidence of fatty liver disease was detected by magnetic resonance spectroscopy in all participants with SBMA in the first group, with an average dome intrahepatic triacylglycerol of 27% (range 6%–66%, ref ≤5.5%). Liver dome magnetic resonance spectroscopy measurements were significantly increased in participants with SBMA in the second group relative to age- and sex-matched controls, with average disease and male control measurements of 17% and 3%, respectively. Liver biopsies were consistent with simple steatosis in 2 participants and nonalcoholic steatohepatitis in 2 others.Conclusions:We observed evidence of nonalcoholic liver disease in nearly all of the participants with SBMA evaluated. These observations expand the phenotypic spectrum of the disease and provide a potential biomarker that can be monitored in future studies.

The lack of standardization of tissue handling and processing hinders the development and validation of new biomarkers in research and clinical settings. We compared the histomorphology and the quality and quantity of biomolecules in paraffin-embedded mouse tissues, followed by fixation with neutral buffered formalin (NBF), 70% ethanol, and buffered ethanol (BE70) fixative. The quality of the histomorphology and immunohistochemistry in BE70 was relatively time-independent, whereas those in NBF rapidly decreased after 1 week of fixation. Protein recovered from tissue fixed in 70% ethanol and BE70 was compatible with Western blot and protein array using AKT and GAPDH antibodies, regardless of the fixation time. In addition, the quality and quantity of RNA extracted from tissue in ethanol-based fixative showed minimal changes from 4 hr to 6 months, whereas NBF had a dramatic detrimental change in RNA quality after 1 week of fixation. Furthermore, ethanol-based fixative offers a superior DNA template for PCR amplification–based molecular assays than NBF. In conclusion, coagulative, ethanol-based fixatives show a broader time spectrum than the aldehyde crosslinking fixative NBF in their histomorphological features and the quantity and quality of the biomolecules from paraffin-embedded tissue, and they may facilitate the use of fixative-fixed paraffin-embedded tissues in research and clinical laboratories, avoiding overfixation.

Background The interaction of vascular endothelial growth factor-C (VEGF-C)/VEGF-D/VEGF receptor-3 is considered to be a major driver of lymphangiogenesis, however the mechanism of this process remains unclear. We aimed to investigate the possible lymphangiogenic significance of synaptonemal complex protein 3 (SCP3) in non-small cell lung cancer (NSCLC). Methods The expression of SCP3, VEGF-C, and VEGF-D were measured and examined a correlation between SCP3 and VEGF-C or VEGF-D in various human lung cancer cell lines. Subsequently, we assessed SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D expression in archival tumor tissues from 89 NSCLC patients with lymph node (LN) metastasis by combined immunohistochemistry with quantitative digital image analysis. Results Positive correlations between SCP3 and VEGF-C expression (R 2 = 0.743) and VEGF-D expression (R 2 = 0.932) were detected in various human lung cancer cell lines. The high expression of SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D were detected in 24 (27.0%), 22 (24.7%), 27 (30.3%), 27 (30.3%), and 24 cases (27.0%), respectively. Notably, SCP3 positively correlated with VEGF-C and VEGF-D expression (for both, P

Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin-fixed, paraffin-embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neoplasia grade 3, and cancer using a bead-based microarray covering 1,500 CpG sites in over 800 genes. Methylation levels in individual CpG sites were compared using a t-test, and results were summarized by computing p-values. A total of 12 candidate genes (ADCYAP1, ASCL1, ATP10, CADM1, DCC, DBC1, HS3ST2, MOS, MYOD1, SOX1, SOX17 and TMEFF2) identified by DNA methylation profiling, plus an additional three genes identified from the literature (EPB41L3, MAL and miR-124) were chosen for validation in an independent set of 167 liquid-based cytology specimens using pyrosequencing and targeted, next-generation bisulfite sequencing. Of the 15 candidate gene markers, 10 had an area under the curve (AUC) of ≥ 0.75 for discrimination of high grade squamous intraepithelial lesions or worse (HSIL+) from