Histomorphological evaluation, cell proliferation and endothelial immunostaining in oral and maxillofacial myofibroblastic lesions

Background: Myofibroblasts (MF) are mesenchymal cells with features of both fibroblasts and smooth muscle cells. Although these are usually reactive cells, they can lead to myofibroblastic tumors that may share clinical and histomorphological characteristics but with different prognosis. The aim of this study is to perform a histomorphological evaluation as well as to compare and evaluate two different cell proliferation immunomarkers and two endothelial markers in a group of oral and maxillofacial myofibroblastic lesions (MFL). Material and methods: Cross-sectional and retrospective study. Demographic, clinical, histomorphological and immunohistochemical characteristics of 39 cases of MFL were analyzed. Immunohistochemical reactions were performed with the Ki67, MCM2, CD34 and CD105 antibodies. Kruskal-Wallis test and Spearman correlation analysis were used. Results: Four cases of nodular fasciitis (NF), 18 myofibromas (My), 6 desmoplastic fibromas (DF), 7 inflammatory myofibroblastic tumors (IMT) and 4 myofibroblastic sarcomas (MFS) were studied. There were twenty women (51.2%); the median age was 13 [Q1-Q3: 8-24] years and most cases occurred in the mandible (48.7%). A statistically significant difference with MCM2 immunostaining (p=0.0221) was observed between the MFL; furthermore, a correlation between CD34 and CD105 immunostaining in NF (p <0.0001) and IMT (p=0.0408), between MCM2 and CD34 in IMT (p=0.0362) and between MCM2 and CD105 in MFS (p <0001) were found. Conclusions: MCM2 immunostaining could assess more clearly the cell growth fraction in MFL. The correlation between MCM2 and CD34 in IMT and between MCM2 and CD105 in MFS are indicative of the high activity of these lesions. These results emphasize the importance of the studied immunohistochemistry markers as possible tools for a better characterization of some of the MFL. (AU)

Histomorphological evaluation, cell proliferation and endothelial immunostaining in oral and maxillofacial myofibroblastic lesions.

BACKGROUND: Myofibroblasts (MF) are mesenchymal cells with features of both fibroblasts and smooth muscle cells. Although these are usually reactive cells, they can lead to myofibroblastic tumors that may share clinical and histomorphological characteristics but with different prognosis. The aim of this study is to perform a histomorphological evaluation as well as to compare and evaluate two different cell proliferation immunomarkers and two endothelial markers in a group of oral and maxillofacial myofibroblastic lesions (MFL). MATERIAL AND METHODS: Cross-sectional and retrospective study. Demographic, clinical, histomorphological and immunohistochemical characteristics of 39 cases of MFL were analyzed. Immunohistochemical reactions were performed with the Ki67, MCM2, CD34 and CD105 antibodies. Kruskal-Wallis test and Spearman correlation analysis were used. RESULTS: Four cases of nodular fasciitis (NF), 18 myofibromas (My), 6 desmoplastic fibromas (DF), 7 inflammatory myofibroblastic tumors (IMT) and 4 myofibroblastic sarcomas (MFS) were studied. There were twenty women (51.2%); the median age was 13 [Q1-Q3: 8-24] years and most cases occurred in the mandible (48.7%). A statistically significant difference with MCM2 immunostaining (p=0.0221) was observed between the MFL; furthermore, a correlation between CD34 and CD105 immunostaining in NF (p <0.0001) and IMT (p=0.0408), between MCM2 and CD34 in IMT (p=0.0362) and between MCM2 and CD105 in MFS (p <0001) were found. CONCLUSIONS: MCM2 immunostaining could assess more clearly the cell growth fraction in MFL. The correlation between MCM2 and CD34 in IMT and between MCM2 and CD105 in MFS are indicative of the high activity of these lesions. These results emphasize the importance of the studied immunohistochemistry markers as possible tools for a better characterization of some of the MFL.

Uterine Inflammatory Myofibroblastic Neoplasms With Aggressive Behavior, Including an Epithelioid Inflammatory Myofibroblastic Sarcoma: A Clinicopathologic Study of 9 Cases.

The experience with uterine inflammatory myofibroblastic neoplasms with an unfavorable outcome is limited. We present the clinicopathologic features of 9 such cases, including 8 inflammatory myofibroblastic tumors (IMTs) and 1 epithelioid inflammatory myofibroblastic sarcoma (EIMS). Median patient age for the IMT group was 50.5 years; the patient with EIMS was 43 years old. Patients presented with abnormal uterine bleeding, presumed fibroids, pelvic pain, arthralgia and low-grade fever, as well as an incidental finding. Median tumor size for the IMTs was 8.5 cm. The borders were either infiltrative or well-circumscribed. Histologically, IMTs were purely fascicular or myxoid or showed predominance of one or the other pattern. Seven tumors were spindled, and 1 was both spindled and epithelioid. Tumors had variable nuclear atypia, ranging from grade 1 to 3. All tumors had an inflammatory infiltrate-predominantly lymphocytic, majority had necrosis (62.5%) and none had lymphovascular invasion. 7/8 (87.5%) tumors were positive for ALK-1 by immunohistochemistry (IHC). One tumor was negative for ALK-1 by IHC but was positive for ALK fusion by fluorescence in situ hybridization and had TNS1-ALK fusion by next-generation sequencing (NGS). Three other tumors with NGS testing showed one of the following ALK-fusion partners: FN1, DCTN1, and IGFBP5. The EIMS had infiltrative borders, myxoid and hyalinized patterns, epithelioid cells, and no lymphovascular invasion. This tumor was ALK-1 positive by IHC, had ALK rearrangement by fluorescence in situ hybridization and RANBP2-ALK fusion by NGS. Extrauterine disease at time of diagnosis was noted in 2/8 (25%) of IMTs, and in the single case of EIMS. Seven patients had surgery as primary treatment, 1 patient had neoadjuvant chemotherapy and 1 patient declined treatment. Patients with recurrence were treated with a combination of chemotherapy, targeted therapy, radiotherapy or hormonal therapy. Most patients (71.4%) recurred within 24 months (mos). Two thirds of patients were alive with disease at last follow up (mean 43.6 mos). The patient with EIMS was alive with disease at 22 mos. IMT referral cases were initially diagnosed as smooth muscle tumors in 87.5% of cases; while the EIMS was diagnosed as high-grade endometrial stromal sarcoma. Lack of consideration of IMT in the differential diagnosis of smooth muscle tumors with myxoid features can result in misdiagnosis and under-utilization of targeted therapy in these patients.

Nodular fasciitis: a comprehensive, time-correlated investigation of 17 cases.

The self-limited nature of nodular fasciitis (NF) is well-known but its precise mechanism has not yet been clarified. We observed that "young" NF (preoperative duration <1 month) consistently contains a higher percentage (~80%) of USP6 break-apart FISH signals than "old" NF (preoperative duration >3 months) (~20%). Thus, we hypothesized that our original observation may reflect a connection with the self-limited nature of NF. Seventeen cases with reliable data concerning the onset were selected, thus approximating the lifetime of each tumor. Besides the USP6 interphase FISH examination, we also checked the most common MYH9-USP6 fusion using RT-PCR. Because of the known pathways of the tumorigenesis of NF, the mRNA level of USP6, TRAIL, IFN-beta, JAK1, STAT1, STAT3, JUN, and CDKN2A was measured using qRT-PCR. Regarding proteins, USP6, p16, p27, TRAIL, and IFN-beta were examined using immunohistochemistry. Targeted gene panel next-generation sequencing (NGS) of three cases was additionally performed. We found a strong negative correlation (p = 0.000) between the lifetime and percentage of USP6 break-apart signals and a strong positive relationship (p = 0.000) between USP6 break-apart signals and mitotic counts. Results of immunostainings, along with qRT-PCR results, favored the previously-suggested USP6-induced negative feedback mechanism through activation of TRAIL and IFN-beta, likely resulting in apoptosis and senescence of tumor cells harboring USP6 fusions. Targeted-NGS resulted in the detection of several variants, but no additional recurrent changes in the pathogenesis of these tumors. We revealed on a cellular level the USP6-induced negative feedback mechanism. In conclusion, we emphasize that in "old" NF, the percentage of USP6 break-apart FISH signals can be as low as 14-27% which can be very important from a differential diagnostic point of view. We emphasize that a careful examination and interpretation of the NGS data is needed before clinical decision-making on treatment.

Fibroblast and Myofibroblast Subtypes: Single Cell Sequencing.

The stroma constitutes the structural framework of an organ and plays crucial roles in health and following organ damage. The major player of the stroma with respect to extracellular matrix deposition, maintenance, and remodeling is the fibroblast and its activated derivative, the myofibroblast. It has long been recognized that there is considerable variability to the fibroblast phenotype. The recent advent of new single cell "omics" technologies has revolutionized our understanding and appreciation of cellular heterogeneity of fibroblasts been revolutionized. With these tools, the nature and defining characteristics of the cells comprising the stroma is finally being defined not just through a few markers, but by taking a wholistic look at transcriptional programs. It is now apparent that stromal cells are not only transcriptionally diverse, but also functionally, epigenetically, and spatially heterogeneous. Studying populations at single cell resolution has enabled identification of new clusters of cells with unique transcriptional signatures. Whether these clusters truly represent distinct subpopulations or different states of the same population remains to be clarified. In this chapter, we first describe a procedure for purification and preparation of a single cell suspension from tissue samples (in this case the heart) for single cell RNA sequencing. We also describe preparation of high-quality tissue sections for spatial transcriptomics. Secondly, we outline a workflow for computational analysis of single cell RNA sequencing and spatial transcriptomics data, as well as integrating them together, to explore the heterogeneity within fibroblasts/myofibroblasts and identify different subtypes and their locations in the heart.

Senescence and autophagy in usual interstitial pneumonia of different etiology.

Idiopathic pulmonary fibrosis (IPF) is a disease with a dismal prognosis. Currently, the causing agent(s) are poorly understood. Recent data suggest that senescence and autophagy might play a role in its development, as well as changes in metabolism due to hypoxic conditions. In this study, the expression of senescence markers in 23 cases of usual interstitial pneumonia (UIP)/IPF and UIP/chronic autoimmune diseases (UIP/AuD) was investigated. The status of autophagy was evaluated with respect to either antiinflammatory or antihypoxia function. Formalin-fixed paraffin-embedded tissues of UIP were selected for immunohistochemistry with antibodies for p21, p16, and ß-galactosidase (senescence); for LC3, SIRT1, MAP1S, and pAMKα (autophagy); and for LDH and GLUT1 (metabolism). Epithelial cells in cystic remodeled areas of UIP stained for p16 and p21, p16 being more specific compared with p21. Myofibroblasts were negative in all cases. An upregulation of all four autophagy markers was seen not only in epithelia within remodeled areas and proliferating myofibroblasts, but also in bronchial epithelia and pneumocytes. Upregulated autophagy points to a compensatory mechanism for hypoxia; therefore, LDH and GLUT1 were investigated. Their expression was present in epithelia within cystic remodeling and in myofibroblasts. The cells within the remodeled areas stained for cytokeratin 5, but coexpressed TTF1, confirming their origin from basal cells of bronchioles. Within this population, senescent cells arise. Our results indicated that autophagy in UIP very likely helps cells to survive in hypoxic condition. By phagocytosis of cellular debris, they supplement their need for nutrition, and by upregulating LDH and GLUT1, they compensate for local hypoxia.

Inflammatory myofibroblastic tumors associated with the placenta: a series of 9 cases.

Inflammatory myofibroblastic tumors (IMTs) of the uterus are often associated with pregnancy and are delivered with the placenta. We describe the clinical, pathologic, and molecular findings in nine cases of placenta-associated IMT (PaIMT). All the lesions were incidentally discovered at delivery or on placental pathological examination. The maternal age ranged from 21 to 41 (mean = 30.6) years. Eight patients had high-risk pregnancies, and when known, all patients were multigravida. Macroscopically, eight tumors were well defined, ranging in size from 2 to 6 cm present at the maternal surface of the placenta (n = 3) and membranes (n = 4) or separately delivered with the placenta (n = 2). All nine lesions revealed classical IMT morphology with spindle cells associated with a lymphoplasmacytic infiltrate and thin elongated vessels. Five showed decidualization, and five showed coagulative necrosis. All tumors expressed CD10. Of the seven tumors that were anaplastic lymphoma kinase (ALK) positive, six were confirmed to have an ALK rearrangement by fluorescence in situ hybridization (FISH), whereas one failed FISH testing. Fusions included TIMP3-ALK (n = 3), THBS1-ALK (n = 2), and a novel SYN3-ALK fusion (n = 1). Clinical follow-up was available in three patients, with no recurrence reported. There appears to be an increased frequency of uterine IMTs in pregnancy and associated with the placenta. No PaIMT has behaved aggressively, although follow-up has been quite limited. This may speak to a specific behavior of these tumors when associated with pregnancy.

MicroRNA­375 prevents TGF­ß­dependent transdifferentiation of lung fibroblasts via the MAP2K6/P38 pathway.

Transdifferentiation of lung fibroblasts to myofibroblasts is a crucial pathophysiological process in pulmonary fibrosis. MicroRNA­375 (miR­375) was initially identified as a tumor­suppressive factor, and its expression was negatively associated with the severity of lung cancer; however, its role and potential mechanism in myofibroblast transdifferentiation and pulmonary fibrosis remain unclear. In the present study, human lung fibroblasts were stimulated with transforming growth factor­ß (TGF­ß) to induce myofibroblast transdifferentiation. A mimic and inhibitor of miR­375, and their negative controls, were used to overexpress or suppress miR­375 in lung fibroblasts, respectively. The mRNA expression levels of fibrotic markers, and protein expression of α­smooth muscle actin and periostin, were subsequently detected by reverse transcription­quantitative PCR and western blotting, to assess myofibroblast transdifferentiation. miR­375 was markedly upregulated in human lung fibroblasts after TGF­ß stimulation. The miR­375 mimic alleviated, whereas the miR­375 inhibitor aggravated TGF­ß­dependent transdifferentiation of lung fibroblasts. Mechanistically, miR­375 prevented myofibroblast transdifferentiation and collagen synthesis by blocking the P38 mitogen­activated protein kinases (P38) pathway, and P38 suppression abrogated the deleterious effect of the miR­375 inhibitor on myofibroblast transdifferentiation. Furthermore, the present study revealed that mitogen­activated protein kinase kinase 6 was involved in P38 inactivation by miR­375. In conclusion, miR­375 was implicated in modulating TGF­ß­dependent transdifferentiation of lung fibroblasts, and targeting miR­375 expression may help to develop therapeutic approaches for treating pulmonary fibrosis.

Prognostic relevance of the loss of stromal CD34 positive fibroblasts in invasive lobular carcinoma of the breast.

CD34+ fibroblasts are constitutive stromal components of virtually all organs, including the mammary stroma, being involved in matrix synthesis, antigen presentation, and tumor-associated stromal remodeling. The most common subtype of invasive breast carcinoma, invasive carcinoma of no special type (IBC-NST), is known for its stromal loss of CD34+ fibroblasts while acquiring alpha smooth muscle actin-positive (α-SMA+) myofibroblasts, i.e., cancer-associated fibroblasts (CAF), whereas invasive lobular carcinoma (ILC) displays partial preservation of CD34+ fibroblasts. The aim of this study was to evaluate the prognostic relevance of stromal CD34+ fibroblasts and α-SMA+ myofibroblasts in an extended collection of ILC. A total of 133 cases of ILC, primarily resected between 1996 and 2004 at University Hospital Marburg, were examined semiquantitatively for stromal content of CD34+ fibroblasts and α-SMA+ myofibroblasts. Partial preservation of CD34+ fibroblasts in the tumor stroma of ILC was confirmed. Absence of CD34+ fibroblasts in the tumor stroma significantly correlated with the presence of α-SMA+ myofibroblasts (p = 0.010), positive lymph node status (p = 0.004), and pN stage (p = 0.006). Stromal loss of CD34+ fibroblasts was significantly associated with lower overall and disease-free survival rates (p = 0.012 and 0.013, respectively). Multivariate analysis adjusted for pT and pN stage revealed stromal loss of CD34+ fibroblasts as independent prognostic parameter (p = 0.05). To our knowledge, this is the first report defining prognostically relevant stromal subtypes of ILC with long-term follow-up. Future research targeting the potential diagnostic and therapeutic implications of CD34+ fibroblasts and CAF in breast cancer, especially ILC, is a promising field of interest.

Loss of Family with Sequence Similarity 13, Member A Exacerbates Pulmonary Fibrosis Potentially by Promoting Epithelial to Mesenchymal Transition.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with poor prognosis due to limited clinical treatment options. IPF is characterized by the augmented deposition of extracellular matrix driven by myofibroblasts, and the epithelial-mesenchymal transition (EMT) has been known to play an essential role in the mechanism of pulmonary fibrosis. Previous genome-wide association study identified Fam13a as one of genes that showed genetic link with IPF and chronic obstructive pulmonary disease. Here, we analyzed the role of Fam13a in the pathogenesis of pulmonary fibrosis using Fam13a-deficient mice. We found that Fam13a was down-regulated in mouse lungs of bleomycin-induced pulmonary fibrosis model. Of note, genetic deletion of Fam13a exacerbated the lung fibrosis induced by bleomycin in association with enhanced EMT in mice. Moreover, silencing of Fam13a accelerated EMT induced by TGF-ß and TNF-α in alveolar epithelial cells, accompanied by increased active ß-catenin and its nuclear accumulation. Our data revealed a crucial role of Fam13a in the development of pulmonary fibrosis potentially through inhibiting EMT, and thus Fam13a has a therapeutic potential in the treatment of IPF.

Living myofibroblast-silicon composites for probing electrical coupling in cardiac systems.

Traditional bioelectronics, primarily comprised of nonliving synthetic materials, lack cellular behaviors such as adaptability and motility. This shortcoming results in mechanically invasive devices and nonnatural signal transduction across cells and tissues. Moreover, resolving heterocellular electrical communication in vivo is extremely limited due to the invasiveness of traditional interconnected electrical probes. In this paper, we present a cell-silicon hybrid that integrates native cellular behavior (e.g., gap junction formation and biosignal processing) with nongenetically enabled photosensitivity. This hybrid configuration allows interconnect-free cellular modulation with subcellular spatial resolution for bioelectric studies. Specifically, we hybridize cardiac myofibroblasts with silicon nanowires and use these engineered hybrids to synchronize the electrical activity of cardiomyocytes, studying heterocellular bioelectric coupling in vitro. Thereafter, we inject the engineered myofibroblasts into heart tissues and show their ability to seamlessly integrate into contractile tissues in vivo. Finally, we apply local photostimulation with high cell specificity to tackle a long-standing debate regarding the existence of myofibroblast-cardiomyocyte electrical coupling in vivo.

Quantification of stromal reaction in breast carcinoma and its correlation with tumor grade and free progression survival.

Cancer progression results from a complex interplay between tumor cells and the extracellular milieu. In breast carcinoma, the stromal microenvironment has been suggested to play a major role in promoting tumor growth, progression, and invasion. The stroma of 154 resected specimens of invasive breast carcinoma of no special type was quantified using a digital image analyzer. Statistical analyses were performed between the quantity of stroma and survival, as well as between progression-free survival and clinicopathological data. Levels of myofibroblastic stroma varied from 0-46%, with a median of 15.1% and a standard deviation of 7.5. The myofibroblastic stromal reaction was statistically greater in grade 2 and 3 tumors (p = 0.029). Furthermore, there was a trend for worse progression-free survival in the group of node-negative tumors with strong smooth-muscle actin stromal expression (Log rank = 0.075). The present study demonstrates that the myofibroblastic reaction of breast invasive carcinoma of no special type is not merely a passive reaction, but seems to be an integral part of the neoplastic process by facilitating tumor progression and invasion. Additional, larger studies on mechanisms of stromal change are needed and may potentially lead to novel treatments.

Increased mesenchymal podoplanin expression is associated with calcification in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (AR + f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in AR + f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.

Protease-activated receptors are potential regulators in the development of arterial endofibrosis in high-performance athletes.

OBJECTIVE: High-performance athletes can develop symptomatic arterial flow restriction during exercise caused by endofibrosis. The pathogenesis is poorly understood; however, coagulation enzymes, such as tissue factor (TF) and coagulation factor Xa, might contribute to the fibrotic process, which is mainly regulated through activation of protease-activated receptors (PARs). Therefore, the aim of this explorative study was to evaluate the presence of coagulation factors and PARs in endofibrotic tissue, which might be indicative of their potential role in the natural development of endofibrosis. METHODS: External iliac arterial specimens with endofibrosis (n = 19) were collected during surgical interventions. As control, arterial segments of the external iliac artery (n = 20) were collected post mortem from individuals with no medical history of cardiovascular disease who donated their body to medical science. Arteries were paraffinized and cut in tissue sections for immunohistochemical analysis. Positive staining within lesions was determined with ImageJ software (National Institutes of Health, Bethesda, Md). RESULTS: Endofibrotic segments contained a neointima, causing intraluminal stenosis, which was highly positive for collagen (+150%; P < .01) and elastin (+148%; P < .01) in comparison with controls. Intriguingly, endofibrosis was not limited to the intima because collagen (+213%) and elastin (+215%) were also significantly elevated in the media layer of endofibrotic segments. These findings were accompanied by significantly increased α-smooth muscle actin-positive cells, morphologically compatible with the presence of myofibroblasts. In addition, PAR1 and PAR4 and the membrane receptor TF were increased as well as coagulation factor X. CONCLUSIONS: We showed that myofibroblasts and the accompanying collagen and elastin synthesis might be key factors in the development of endofibrosis. The special association with increased presence of PARs, factor X, and TF suggests that protease-mediated cell signaling could be a contributing component in the mechanisms leading to endofibrosis.

EWSR1-SMAD3-rearranged Fibroblastic Tumor: An Emerging Entity in an Increasingly More Complex Group of Fibroblastic/Myofibroblastic Neoplasms.

Three cases of superficial acral fibroblastic spindle cell neoplasms with EWSR1-SMAD3 fusion have been recently reported. Their differential diagnosis is broad, primarily comprising rare tumors from the fibroblastic/myofibroblastic category. The aim of this report is to present 4 new cases of this entity and to discuss the appropriate differential diagnosis. Also, as the ERG antibody seems to be a characteristic marker for these tumors, we analyzed ERG immunostaining characteristics in potential mimics of this entity. All cases in our cohort occurred in women aged 5 to 68 years (mean, 36.5 y). Two were located on the hand, 1 on foot, and the last case arose on the calf. The tumor size ranged from 1 to 1.5 cm in the greatest dimension, with a mean size of 1.2 cm. Except for one recent case, follow-up was available, ranging from 7 to 18 years (mean, 11.7 y), with a recurrence noted in 1 case after 10 years. All tumors were subcutaneous and showed 2 main components. One consisted of bland, spindled cells with elongated nuclei which were round when observed on the cross-section. These cells mostly grew in relatively hypercellular, well-organized, and intersecting fascicles. The second component was prominently hyalinized and paucicellular, but lacked calcifications. Both components showed either a distinct zonation pattern, or they were randomly intermingled with each other. In all 3 analyzable tumors, next-generation sequencing showed EWSR1-SMAD3 gene fusion in each case. By fluorescence in situ hybridization, one tested case also revealed unbalanced rearrangement of the EWSR1 gene. All 4 cases showed strong, diffuse nuclear expression of ERG, whereas none of the mimics stained with this antibody except for weak to moderate staining in calcifying aponeurotic fibromas (9/10 cases). Two tumors showed focal weak to moderate expression of SAT-B2. The 4 herein presented cases further broaden the clinicopathologic spectrum of tumors with EWSR1-SMAD3 gene fusion. They also confirm that they represent a novel entity for which we propose the name EWSR1-SMAD3-rearranged fibroblastic Tumor. Our study also proves that in the context of fibroblastic/myofibroblastic tumors, ERG immunohistochemistry is a relatively specific marker for these neoplasms.

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts.

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-ß1 (TGF-ß1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-ß1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-ß1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

Inflammatory Myofibroblastic Tumor Associated With the Placenta: Short Tandem Repeat Genotyping Confirms Uterine Site of Origin.

Inflammatory myofibroblastic tumor (IMT) is a neoplasm of intermediate malignant potential that only rarely involves the gynecologic tract. Several cases of IMT arising in various locations including the lung, bladder, trachea, and breast in association with pregnancy have been reported in the literature, and 3 cases involving the placenta have been previously described. We report 2 cases of IMT identified in association with pregnancy; the first was an intrauterine mass delivered entirely separate from the placenta and fetus, and the second was an incidental mass identified within the placental parenchyma following delivery. Short tandem repeat genotyping was used to compare tissue from the tumor and the placenta for both cases. Both tumors were determined to be of maternal origin, confirming that uterine IMTs may present within the placenta or as a separate mass following delivery. This demonstrates the utility of short tandem repeat genotyping in determining the origin of tumors presenting in association with the placenta.

Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4.

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

Identification of a myofibroblast-specific expression signature in skin wounds.

After skin injury fibroblasts migrate into the wound and transform into contractile, extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury, their number reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. Most of these genes were linked with the extracellular region and cell periphery including genes encoding for extracellular matrix proteins. A unique panel of core matrisome and matrisome-associated genes was differentially expressed in myofibroblasts and several genes not yet known to be linked to myofibroblast-mediated wound healing were found (e.g. Col24a1, Podnl1, Bvcan, Tinagl1, Thbs3, Adamts16, Adamts19, Cxcl's, Ccl's). In addition, a complex network of G protein-coupled signaling events was regulated in myofibroblasts (e.g. Adcy1, Plbc4, Gnas). Hence, this first characterization of a myofibroblast-specific expression profile at the peak of in situ granulation tissue formation provides important insights into novel target genes that may control excessive ECM deposition during fibrotic reactions.

p70S6K activation promotes the transdifferentiation of fibroblasts to myofibroblasts in pterygium tissue growth on the cornea.

OBJECTIVE: To investigate the role of p70S6K in the transdifferentiation of fibroblasts to myofibroblasts in pterygium tissue growth on the cornea. RESULTS: Quantitative real-time PCR and immunohistochemistry showed that p70S6K expression was higher in pterygium tissues than in normal conjunctival tissues. Higher p70S6K RNA expression levels were correlated with higher pterygium grades. Additionally, western blot analysis revealed that phosphorylated (activated) p70S6K (p-p70S6K) expression was significantly correlated with α-smooth muscle actin (α-SMA, a hallmark of transdifferentiation) expression in cultured human pterygium fibroblasts (HPFs). Furthermore, p70S6K knockdown and the specific mTOR inhibitor rapamycin decreased the expression levels of p-p70S6K and α-SMA in cultured fibroblasts from grade T3 pterygium. CONCLUSIONS: p70S6K activation promotes the transdifferentiation of pterygium fibroblasts to myofibroblasts. Thus, targeting p70S6K may be a useful strategy in the management of pterygium.