Predicting HPV association using deep learning and regular H&E stains allows granular stratification of oropharyngeal cancer patients.

Human Papilloma Virus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) represents an OPSCC subgroup with an overall good prognosis with a rising incidence in Western countries. Multiple lines of evidence suggest that HPV-associated tumors are not a homogeneous tumor entity, underlining the need for accurate prognostic biomarkers. In this retrospective, multi-institutional study involving 906 patients from four centers and one database, we developed a deep learning algorithm (OPSCCnet), to analyze standard H&E stains for the calculation of a patient-level score associated with prognosis, comparing it to combined HPV-DNA and p16-status. When comparing OPSCCnet to HPV-status, the algorithm showed a good overall performance with a mean area under the receiver operator curve (AUROC) = 0.83 (95% CI = 0.77-0.9) for the test cohort (n = 639), which could be increased to AUROC = 0.88 by filtering cases using a fixed threshold on the variance of the probability of the HPV-positive class - a potential surrogate marker of HPV-heterogeneity. OPSCCnet could be used as a screening tool, outperforming gold standard HPV testing (OPSCCnet: five-year survival rate: 96% [95% CI = 90-100%]; HPV testing: five-year survival rate: 80% [95% CI = 71-90%]). This could be confirmed using a multivariate analysis of a three-tier threshold (OPSCCnet: high HR = 0.15 [95% CI = 0.05-0.44], intermediate HR = 0.58 [95% CI = 0.34-0.98] p = 0.043, Cox proportional hazards model, n = 211; HPV testing: HR = 0.29 [95% CI = 0.15-0.54] p < 0.001, Cox proportional hazards model, n = 211). Collectively, our findings indicate that by analyzing standard gigapixel hematoxylin and eosin (H&E) histological whole-slide images, OPSCCnet demonstrated superior performance over p16/HPV-DNA testing in various clinical scenarios, particularly in accurately stratifying these patients.

Sex-specific prognostic effect of CD66b-positive tumor-infiltrating neutrophils (TANs) in gastric and esophageal adenocarcinoma.

BACKGROUND: Tumor-associated neutrophils (TANs) have recently been identified as a relevant component of the tumor microenvironment (TME) in solid tumors. Within the TME TANs mediate either tumor-promoting or tumor-inhibiting activities. So far, their prognostic relevance remains to be determined. This study aims to evaluate the prognostic relevance of TANs in different molecular subtypes of gastric and esophageal adenocarcinoma. METHODS: We analyzed a total of 1118 Caucasian patients divided into gastric adenocarcinoma (n = 458) and esophageal adenocarcinoma cohort (n = 660) of primarily resected and neoadjuvant-treated individuals. The amount of CD66b + TANs in the tumor stroma was determined using quantitative image analysis and correlated to both molecular, as well as clinical data. RESULTS: An accumulation of TANs in the tumor stroma of gastric carcinomas was associated to a significant favorable prognosis (p = 0.026). A subgroup analysis showed that this effect was primarily related to the molecular chromosomal instable subtype (CIN) of gastric carcinomas (p = 0.010). This was only observed in female patients (p = 0.014) but not in male patients (p = 0.315). The same sex-specific effect could be confirmed in adenocarcinomas of the esophagus (p = 0.027), as well as in female individuals after receiving neoadjuvant therapy (p = 0.034). CONCLUSIONS: Together, we show a sex-specific prognostic effect of TANs in gastric cancer within a Caucasian cohort. For the first time, we showed that this sex-specific prognostic effect of TANs can also be seen in esophageal cancer.

Deep Learning Predicts HPV Association in Oropharyngeal Squamous Cell Carcinomas and Identifies Patients with a Favorable Prognosis Using Regular H&E Stains.

PURPOSE: Human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) is tumorigenic and has been associated with a favorable prognosis compared with OPSCC caused by tobacco, alcohol, and other carcinogens. Meanwhile, machine learning has evolved as a powerful tool to predict molecular and cellular alterations of medical images of various sources. EXPERIMENTAL DESIGN: We generated a deep learning-based HPV prediction score (HPV-ps) on regular hematoxylin and eosin (H&E) stains and assessed its performance to predict HPV association using 273 patients from two different sites (OPSCC; Giessen, n = 163; Cologne, n = 110). Then, the prognostic relevance in a total of 594 patients (Giessen, Cologne, HNSCC TCGA) was evaluated. In addition, we investigated whether four board-certified pathologists could identify HPV association (n = 152) and compared the results to the classifier. RESULTS: Although pathologists were able to diagnose HPV association from H&E-stained slides (AUC = 0.74, median of four observers), the interrater reliability was minimal (Light Kappa = 0.37; P = 0.129), as compared with AUC = 0.8 using the HPV-ps within two independent cohorts (n = 273). The HPV-ps identified individuals with a favorable prognosis in a total of 594 patients from three cohorts (Giessen, OPSCC, HR = 0.55, P < 0.0001; Cologne, OPSCC, HR = 0.44, P = 0.0027; TCGA, non-OPSCC head and neck, HR = 0.69, P = 0.0073). Interestingly, the HPV-ps further stratified patients when combined with p16 status (Giessen, HR = 0.06, P < 0.0001; Cologne, HR = 0.3, P = 0.046). CONCLUSIONS: Detection of HPV association in OPSCC using deep learning with help of regular H&E stains may either be used as a single biomarker, or in combination with p16 status, to identify patients with OPSCC with a favorable prognosis, potentially outperforming combined HPV-DNA/p16 status as a biomarker for patient stratification.

Integrative Analysis of Pleomorphic Dermal Sarcomas Reveals Fibroblastic Differentiation and Susceptibility to Immunotherapy.

PURPOSE: Pleomorphic dermal sarcoma (PDS) is a rare malignant cutaneous tumor with an unknown cell of origin. Locally defined tumors can be treated by curative excisions, whereas advanced stages of the disease are difficult to treat, using standard regimens. EXPERIMENTAL DESIGN: We performed whole-exome sequencing on a cohort of 28 individuals and corresponding transcriptomic analysis on 21 patients, as well as quantitative IHC image analysis on 27 patients. RESULTS: PDS exhibits a universally high mutational load (42.7 mutations/mega base) with an inflamed, immunogenic tumor microenvironment. Three cases of PDS showed response to immune checkpoint blockade. Local mutation rate variation together with mRNA expression data demonstrate that PDS form a distinct entity, with PDGFRB as a lineage marker. In addition, we found that PDS is of mesenchymal, fibroblastic differentiation. CONCLUSIONS: PDS is of fibroblastic differentiation and exhibits a strong susceptibility to immunotherapy, including a high mutational burden and an inflamed tumor microenvironment.

Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR-34a in a hyperoxia-based mouse model of BPD, where miR-34a expression was markedly increased in platelet-derived growth factor receptor (PDGFR)α-expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR-34a; and inducible, conditional deletion of miR-34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia-induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR-34a target, and using a target site blocker in vivo, the miR-34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR-34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology-relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR-34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

A novel mouse Cre-driver line targeting Perilipin 2-expressing cells in the neonatal lung.

Pulmonary diseases such as chronic obstructive pulmonary disease, lung fibrosis, and bronchopulmonary dysplasia are characterized by the destruction or malformation of the alveolar regions of the lung. The underlying pathomechanisms at play are an area of intense interest since these mechanisms may reveal pathways suitable for interventions to drive reparative processes. Lipid-laden fibroblasts (lipofibroblasts) express the Perilipin 2 (Plin2) gene-product, PLIN2, commonly called adipose-differentiation related protein (ADRP). These cells are also thought to play a role in alveolarization and repair after injury to the alveolus. Progress in defining the functional contribution of lipofibroblasts to alveolar generation and repair is hampered by a lack of in vivo tools. The present study reports the generation of an inducible mouse Cre-driver line to target cells of the ADRP lineage. Robust Cre-mediated recombination in this mouse line was detected in mesenchymal cells of the postnatal lung, and in additional organs including the heart, liver, and spleen. The generation and validation of this valuable new tool to genetically target, manipulate, and trace cells of the ADRP lineage is critical for assessing the functional contribution of lipofibroblasts to lung development and repair.

Origin and characterization of alpha smooth muscle actin-positive cells during murine lung development.

ACTA2 expression identifies pulmonary airway and vascular smooth muscle cells (SMCs) as well as alveolar myofibroblasts (MYF). Mesenchymal progenitors expressing fibroblast growth factor 10 (Fgf10), Wilms tumor 1 (Wt1), or glioma-associated oncogene 1 (Gli1) contribute to SMC formation from early stages of lung development. However, their respective contribution and specificity to the SMC and/or alveolar MYF lineages remain controversial. In addition, the contribution of mesenchymal cells undergoing active WNT signaling remains unknown. Using Fgf10CreERT2 , Wt1CreERT2 , Gli1CreERT2 , and Axin2CreERT2 inducible driver lines in combination with a tdTomatoflox reporter line, the respective differentiation of each pool of labeled progenitor cells along the SMC and alveolar MYF lineages was quantified. The results revealed that while FGF10+ and WT1+ cells show a minor contribution to the SMC lineage, GLI1+ and AXIN2+ cells significantly contribute to both the SMC and alveolar MYF lineages, but with limited specificity. Lineage tracing using the Acta2-CreERT2 transgenic line showed that ACTA2+ cells labeled at embryonic day (E)11.5 do not expand significantly to give rise to new SMCs at E18.5. However, ACTA2+ cells labeled at E15.5 give rise to the majority (85%-97%) of the SMCs in the lung at E18.5 as well as alveolar MYF progenitors in the lung parenchyma. Fluorescence-activated cell sorting-based isolation of different subpopulations of ACTA2+ lineage-traced cells followed by gene arrays, identified transcriptomic signatures for alveolar MYF progenitors versus airway and vascular SMCs at E18.5. Our results establish a new transcriptional landscape for further experiments addressing the function of signaling pathways in the formation of different subpopulations of ACTA2+ cells. Stem Cells 2017;35:1566-1578.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.

Fgf10 deficiency is causative for lethality in a mouse model of bronchopulmonary dysplasia.

Inflammation-induced FGF10 protein deficiency is associated with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants characterized by arrested alveolar development. So far, experimental evidence for a direct role of FGF10 in lung disease is lacking. Using the hyperoxia-induced neonatal lung injury as a mouse model of BPD, the impact of Fgf10 deficiency in Fgf10+/- versus Fgf10+/+ pups was investigated. In normoxia, no lethality of Fgf10+/+ or Fgf10+/- pups was observed. By contrast, all Fgf10+/- pups died within 8 days of hyperoxic injury, with lethality starting at day 5, whereas Fgf10+/+ pups were all alive. Lungs of pups from the two genotypes were collected on postnatal day 3 following normoxia or hyperoxia exposure for further analysis. In hyperoxia, Fgf10+/- lungs exhibited increased hypoalveolarization. Analysis by FACS of the Fgf10+/- versus control lungs in normoxia revealed a decreased ratio of alveolar epithelial type II (AECII) cells over total Epcam-positive cells. In addition, gene array analysis indicated reduced AECII and increased AECI transcriptome signatures in isolated AECII cells from Fgf10+/- lungs. Such an imbalance in differentiation is also seen in hyperoxia and is associated with reduced mature surfactant protein B and C expression. Attenuation of the activity of Fgfr2b ligands postnatally in the context of hyperoxia also led to increased lethality with decreased surfactant expression. In summary, decreased Fgf10 mRNA levels lead to congenital lung defects, which are compatible with postnatal survival, but which compromise the ability of the lungs to cope with sub-lethal hyperoxic injury. Fgf10 deficiency affects quantitatively and qualitatively the formation of AECII cells. In addition, Fgfr2b ligands are also important for repair after hyperoxia exposure in neonates. Deficient AECII cells could be an additional complication for patients with BPD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair.

Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(ß4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of ß-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IV-induced lung injury.

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

Attenuating endogenous Fgfr2b ligands during bleomycin-induced lung fibrosis does not compromise murine lung repair.

Fibroblast growth factors (Fgfs) mediate organ repair. Lung epithelial cell overexpression of Fgf10 postbleomycin injury is both protective and therapeutic, characterized by increased survival and attenuated fibrosis. Exogenous administration of FGF7 (palifermin) also showed prophylactic survival benefits in mice. The role of endogenous Fgfr2b ligands on bleomycin-induced lung fibrosis is still elusive. This study reports the expression of endogenous Fgfr2b ligands, receptors, and signaling targets in wild-type mice following bleomycin lung injury. In addition, the impact of attenuating endogenous Fgfr2b-ligands following bleomycin-induced fibrosis was tested by using a doxycycline (dox)-based inducible, soluble, dominant-negative form of the Fgfr2b receptor. Double-transgenic (DTG) Rosa26(rtTA/+);tet(O)solFgfr2b mice were validated for the expression and activity of soluble Fgfr2b (failure to regenerate maxillary incisors, attenuated recombinant FGF7 signal in the lung). As previously reported, no defects in lung morphometry were detected in DTG (+dox) mice exposed from postnatal days (PN) 1 through PN105. Female single-transgenic (STG) and DTG mice were subjected to various levels of bleomycin injury (1.0, 2.0, and 3.0 U/kg). Fgfr2b ligands were attenuated either throughout injury (days 0-11; days 0-28) or during later stages (days 6-28 and 14-28). No significant changes in survival, weight, lung function, confluent areas of fibrosis, or hydroxyproline deposition were detected in DTG mice. These results indicate that endogenous Fgfr2b ligands do not significantly protect against bleomycin injury, nor do they expedite the resolution of bleomycin-induced lung injury in mice.

Fgf10-positive cells represent a progenitor cell population during lung development and postnatally.

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10(iCre) knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1(-) E-Cad(-) Epcam(+) Pro-Spc(+) lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45(-) Cd31(-) Sca-1(+)). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases.