A spatially resolved timeline of the human maternal-fetal interface.

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

Effectiveness of chatbots on COVID vaccine confidence and acceptance in Thailand, Hong Kong, and Singapore.

Chatbots have become an increasingly popular tool in the field of health services and communications. Despite chatbots' significance amid the COVID-19 pandemic, few studies have performed a rigorous evaluation of the effectiveness of chatbots in improving vaccine confidence and acceptance. In Thailand, Hong Kong, and Singapore, from February 11th to June 30th, 2022, we conducted multisite randomised controlled trials (RCT) on 2,045 adult guardians of children and seniors who were unvaccinated or had delayed vaccinations. After a week of using COVID-19 vaccine chatbots, the differences in vaccine confidence and acceptance were compared between the intervention and control groups. Compared to non-users, fewer chatbot users reported decreased confidence in vaccine effectiveness in the Thailand child group [Intervention: 4.3 % vs. Control: 17%, P = 0.023]. However, more chatbot users reported decreased vaccine acceptance [26% vs. 12%, P = 0.028] in Hong Kong child group and decreased vaccine confidence in safety [29% vs. 10%, P = 0.041] in Singapore child group. There was no statistically significant change in vaccine confidence or acceptance in the Hong Kong senior group. Employing the RE-AIM framework, process evaluation indicated strong acceptance and implementation support for vaccine chatbots from stakeholders, with high levels of sustainability and scalability. This multisite, parallel RCT study on vaccine chatbots found mixed success in improving vaccine confidence and acceptance among unvaccinated Asian subpopulations. Further studies that link chatbot usage and real-world vaccine uptake are needed to augment evidence for employing vaccine chatbots to advance vaccine confidence and acceptance.

pSTAT1 Is Activated during the Progression of IgA Nephropathy.

Introduction: IgA nephropathy is the most common primary glomerular disease. Its pathogenesis is still poorly understood. Alterations of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway may play an important role in IgA nephropathy. Methods: We evaluated the clinical features, pathology, and tissue staining for lymphocytes and phosphorylated STAT1 (pSTAT1) in 43 patients with biopsy proven IgA nephropathy. They were followed to determine their disease outcomes. All had biopsy tissue and multiple laboratory measurements to assess their kidney disease progression. Sixteen patients underwent repeat kidney biopsy to further assess their clinical status. Results: The median eGFR at baseline was 61 mL/min/1.73 m2 and the median proteinuria was 2,600 mg/d. The median follow-up was 5 years with an average annual decline in eGFR of 2.25 mL/min/1.73 m2. There was significant inflammation and atrophy seen in the first biopsy, which progressed among those who undertook a 2nd biopsy. Compared to healthy kidney tissue, glomeruli and tubulointerstitium demonstrated increased lymphocyte (CD3+) infiltrates and increased pSTAT1 staining by immunohistochemistry. Increased CD3 (p = 0.001) staining and increased pSTAT1 (p = 0.03) correlated with reduced eGFR levels. In repeat biopsy samples, increasing pSTAT1 staining correlated with loss of eGFR over time (p = 0.02). Conclusion: These findings support the hypothesis that pSTAT1 is activated in IgA nephropathy and may play a role in the progression toward kidney failure.

NFE2L2 Mutations Enhance Radioresistance in Head and Neck Cancer by Modulating Intratumoral Myeloid Cells.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations. SIGNIFICANCE: NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

Waning antibody levels after COVID-19 vaccination with mRNA Comirnaty and inactivated CoronaVac vaccines in blood donors, Hong Kong, April 2020 to October 2021.

The mRNA vaccine Comirnaty and the inactivated vaccine CoronaVac are both available in Hong Kong's COVID-19 vaccination programme. We observed waning antibody levels in 850 fully vaccinated (at least 14 days passed after second dose) blood donors using ELISA and surrogate virus neutralisation test. The Comirnaty-vaccinated group's (n = 593) antibody levels remained over the ELISA and sVNT positive cut-offs within the first 6 months. The CoronaVac-vaccinated group's (n = 257) median antibody levels began to fall below the cut-offs 4 months after vaccination.

Angiotensin-converting enzyme 2 (ACE2) expression increases with age in patients requiring mechanical ventilation.

Mortality due to Covid-19 is highly associated with advanced age, owing in large part to severe lower respiratory tract infection. SARS-CoV-2 utilizes the host ACE2 receptor for infection. Whether ACE2 abundance in the lung contributes to age-associated vulnerability is currently unknown. We set out to characterize the RNA and protein expression profiles of ACE2 in aging human lung in the context of phenotypic parameters likely to affect lung physiology. Examining publicly available RNA sequencing data, we discovered that mechanical ventilation is a critical variable affecting lung ACE2 levels. Therefore, we investigated ACE2 protein abundance in patients either requiring mechanical ventilation or spontaneously breathing. ACE2 distribution and expression were determined in archival lung samples by immunohistochemistry (IHC). Tissues were selected from the specimen inventory at a large teaching hospital collected between 2010-2020. Twelve samples were chosen from patients receiving mechanical ventilation for acute hypoxic respiratory failure (AHRF). Twenty samples were selected from patients not requiring ventilation. We compared samples across age, ranging from 40-83 years old in the ventilated cohort and 14-80 years old in the non-ventilated cohort. Within the alveolated parenchyma, ACE2 expression is predominantly observed in type II pneumocytes (or alveolar type II / AT2 cells) and alveolar macrophages. All 12 samples from our ventilated cohort showed histologic features of diffuse alveolar damage including reactive, proliferating AT2 cells. In these cases, ACE2 was strongly upregulated with age when normalized to lung area (p = 0.004) or cellularity (p = 0.003), associated with prominent expression in AT2 cells. In non-ventilated individuals, AT2 cell reactive changes were not observed and ACE2 expression did not change with age when normalized to lung area (p = 0.231) or cellularity (p = 0.349). In summary, ACE2 expression increases with age in the setting of alveolar damage observed in patients on mechanical ventilation, providing a potential mechanism for higher Covid-19 mortality in the elderly.

PRAME expression in melanocytic proliferations with intermediate histopathologic or spitzoid features.

BACKGROUND: PRAME (PReferentially expressed Antigen in MElanoma) has shown utility in distinguishing melanoma from benign melanocytic lesions, but knowledge of its expression pattern in intermediate melanocytic and spitzoid proliferations is limited. METHODS: Immunohistochemical expression of PRAME was examined in 112 melanocytic proliferations with intermediate histopathologic or spitzoid features. RESULTS: Any intensity of nuclear PRAME staining in at least 60% of lesional melanocytes was determined as the best threshold for diffuse staining in this cohort. Nearly all non-spitzoid melanomas (23/24; 95.8%) demonstrated diffuse PRAME expression. PRAME was completely negative in 95.6% (43/45) of mitotically-active nevi, traumatized nevi, nevi with persistent/recurrent features, and dysplastic nevi. Most Spitz nevi (15/20) and atypical Spitz tumors (10/13) entirely lacked PRAME expression. One Spitz nevus, one atypical Spitz tumor, and one spitzoid melanoma (1/2) demonstrated diffuse PRAME expression. CONCLUSIONS: Although diffuse PRAME expression is generally limited to malignant melanoma, benign Spitz nevi and atypical Spitz tumors can infrequently express diffuse PRAME. PRAME immunohistochemistry can be useful in the evaluation of atypical melanocytic proliferations with intermediate histopathologic features but should be interpreted with caution in the setting of spitzoid neoplasms.

Origins and clonal convergence of gastrointestinal IgE+ B cells in human peanut allergy.

B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE+ and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B lineage cells in food allergy.

The role of Glial cell derived neurotrophic factor in head and neck cancer.

Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRα1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.

Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance.

Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.

Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin.

The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms.Significance: We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. Cancer Discov; 8(10); 1316-31. ©2018 AACR. See related commentary by Pozo et al., p. 1216 This article is highlighted in the In This Issue feature, p. 1195.

Utility of p16 Immunohistochemistry in Evaluating Negative Cervical Biopsies Following High-risk Pap Test Results.

The Lower Anogenital Squamous Terminology (LAST) Standardization Project for human papilloma virus (HPV)-associated lesions specifically recommends the use of p16 immunohistochemistry (IHC) as an adjunct to morphologic assessment of cervical biopsies interpreted as negative or low-grade squamous intraepithelial lesion (LSIL) from patients with prior high-risk Pap test results (high-grade squamous intraepithelial lesion [HSIL], atypical squamous cells cannot exclude HSIL, atypical glandular cells [AGC], or HPV16 atypical squamous cells of undetermined significance [ASC-US]). The impetus for this recommendation is to increase detection of missed high-grade disease. However, the quality of evidence supporting this recommendation was lower than that for the other LAST recommendations addressing improved consistency in the diagnosis of HSIL with the use of p16. A database search spanning 10 years identified 341 cases (encompassing 736 discrete biopsy specimens) interpreted as negative for dysplasia from 330 patients with a prior high-risk Pap result (atypical squamous cells cannot exclude HSIL, HSIL, atypical glandular cells, not otherwise specified [AGC-NOS], atypical endocervical cells--NOS [AEC-NOS], and AEC-favor neoplastic). p16 IHC was performed and detected missed abnormalities in 11/341 (3.2%) cases. The abnormalities corresponded to missed foci of HSIL (cervical intraepithelial neoplasia [CIN] 2) (n=1), SIL-indeterminate grade (n=7), atypical squamous metaplasia (n=2), and LSIL [CIN1]) (n=1). Subsequent histologic follow-up identified HSIL or greater in 6/8 (75%) p16 cases versus 20/79 (25.3%) p16 cases (P=0.0079). p16 IHC performed on biopsies interpreted as negative from patients with prior high-risk Pap test results increased the detection rate of missed SIL. A p16 result also significantly increased the likelihood of HSIL on subsequent biopsy. Although further studies are required to determine what percentage of missed HSIL justifies the additional cost, improved detection of HSIL in high-risk patients may lead to fewer diagnostic procedures and fewer patients lost to follow-up.

CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma.

Tumor-initiating cells (TICs) in squamous cell carcinoma of the head and neck (SCCHN) are best characterized by their surface expression of CD44. Although there is great interest in identifying strategies to target this population, no marker of these cells has been found to be functionally active. Here, we examined the expression of the purported marker of normal human oral epithelial stem cells, CD271. We show that CD271 expression is restricted to a subset of the CD44+ cells. Using xenograft assays, we show that the CD44+CD271+ subpopulation contains the most tumorigenic cells. Loss of CD271 function results in a block in the G2-M phase of the cell cycle and a profound negative impact on the capacity of these cells to initiate tumor formation in vivo. Incubation with recombinant NGF results in enhanced phosphorylation of Erk, providing additional evidence that CD271 is functionally active. Finally, incubation of SCCHN cells with antibody to CD271 results in decreased Erk phosphorylation and decreased tumor formation in vivo. Thus, our data are the first to demonstrate that CD271 more specifically identifies the TIC subpopulation within the CD44+ compartment in SCCHN and that this receptor is a functionally active and targetable molecule.

Human papillomavirus 16 detected in nasopharyngeal carcinomas in white Americans but not in endemic Southern Chinese patients.

BACKGROUND: We evaluated the relationship of human papillomavirus (HPV) and Epstein-Barr virus (EBV) with race in endemic and nonendemic cohorts of patients with nasopharyngeal carcinoma (NPC), and with smoking status in the nonendemic cohort. METHODS: Tissue microarrays (TMAs) were constructed using samples from 86 patients treated in southern China and 108 patients from Stanford, California. TMAs were stained with p16, HPV in situ hybridization (ISH), and EBV ISH. Polymerase chain reaction (PCR) was used to confirm EBV(-) cases and HPV status in p16(+) cases. Survival data was available for the Stanford cohort only. RESULTS: No HPV(+) cases were detected in the Chinese cohort. In the Stanford cohort, 5 of 11 EBV(-) cases harbored HPV-16, 10 of 10 occurred in whites, and 8 of 11 were smokers. Patients with EBV(-) NPC also showed a trend toward worse survival. CONCLUSION: EBV(-) NPC shows an association with the presence of HPV, white race, and smoking. In contrast, EBV(-) NPC shows no association with HPV in the endemic cohort.

An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk.

INTRODUCTION: Outcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples. METHODS: Multiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RTPCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures. RESULTS: RT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention. CONCLUSION: We advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype.

Loss of the p53/p63 target PERP is an early event in oral carcinogenesis and correlates with higher rate of local relapse.

BACKGROUND: PERP is a p53/p63-regulated gene encoding a desmosomal protein that plays a critical role in cell-cell adhesion and tumor suppression. STUDY DESIGN: We evaluated PERP expression in different grades of oral dysplasia (34 cases) and at different stages of invasive squamous cell carcinoma (SCC), and correlated the latter with clinical outcome. A tissue microarray consisting of nondysplastic mucosa, carcinoma in situ, SCC, and nodal metastases from 33 patients with human papilloma virus-negative SCC was stained for PERP and E-cadherin. RESULTS: Complete loss of PERP expression was associated with worse local control in patients with SCC. The 5-year local control rate was 91% for patients with partial PERP loss versus 31% for those with complete loss (P = .01). CONCLUSIONS: This is the first study to show that loss of PERP expression correlates with the transition to SCC and with increased local relapse in patients with oral cavity SCC.

Multiple metabolic alterations exist in mutant PI3K cancers, but only glucose is essential as a nutrient source.

Targeting tumour metabolism is becoming a major new area of pharmaceutical endeavour. Consequently, a systematic search to define whether there are specific energy source dependencies in tumours, and how these might be dictated by upstream driving genetic mutations, is required. The PI3K-AKT-mTOR signalling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival, but has also been associated with metabolic dysregulation. In this study, we sought to define how mutations within PI3KCA may affect the metabolic dependency of a cancer cell, using precisely engineered isogenic cell lines. Studies revealed gene expression signatures in PIK3CA mutant cells indicative of a consistent up-regulation of glycolysis. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. Additional gene expression changes were also observed, suggesting that metabolic pathways other than glycolysis, such as glutaminolysis, were also affected. Nutrient dependency studies revealed that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients. This specific dependence on glucose for growth was further illustrated by studies evaluating the effects of targeted disruption of the glycolytic pathway using siRNA and was also found to be present across a wider panel of cancer cell lines harbouring endogenous PIK3CA mutations. In conclusion, we have found that PIK3CA mutations lead to a shift towards a highly glycolytic phenotype, and that despite suggestions that cancer cells are adept at utilising alternative nutrient sources, PIK3CA mutant cells are not able to compensate for glucose withdrawal. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies and tumour visualisation strategies.