Probes for INS

Chronic microglial activation and associated neuroinflammation are key factors in neurodegenerative diseases including HIV-associated neurocognitive disorders. Colony stimulating factor 1 receptor (CSF1R)-mediated signaling is constitutive in cells of the myeloid lineage, including microglia, promoting cell survival, proliferation, and differentiation. In amyotrophic lateral sclerosis and Alzheimers disease, CSF1R is upregulated. Inhibiting CSF1R signaling in animal models of these diseases improved disease outcomes. In our studies, CNS expression of the CSF1R ligand, colony-stimulating factor 1 (CSF1) was significantly increased in a SIV/macaque model of HIV CNS disease. Using a Nanostring nCounter immune panel, we found CSF1 overexpression was strongly correlated with upregulation of microglial genes involved in antiviral and oxidative stress responses. Using in situ hybridization, we found that CSF1R mRNA was only present in Iba-1 positive microglia. By ELISA and immunostaining with digital image analysis, SIV-infected macaques had significantly higher CSF1R levels in frontal cortex than uninfected macaques (p = 0.018 and p = 0.02, respectively). SIV-infected macaques treated with suppressive ART also had persistently elevated CSF1R similar to untreated SIV-infected macaques. Coordinate upregulation of CSF1 and CSF1R expression implicates this signaling pathway in progressive HIV CNS disease.

Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

The International Endocervical Adenocarcinoma Criteria and Classification was developed to separate endocervical adenocarcinomas (ECAs) into 2 main categories on the basis of morphology such as human papilloma virus-associated (HPVA) and non-human papilloma virus-associated adenocarcinomas. We aimed to improve the diagnostic accuracy of International Endocervical Adenocarcinoma Criteria and Classification by performing a comprehensive immunohistochemical evaluation and constructing objective immunohistochemical-based algorithms for the classification of these tumors. Tissue microarrays were constructed from 297 of 409 cases used to develop the original classification. Immunostains included p16, p53, estrogen receptor (ER), progesterone receptor, androgen receptor, Vimentin, CK7, CK20, HER2, HIK1083, MUC6, CA-IX, SATB2, HNF-1beta, napsin A, PAX8, CDX2, GATA3, p63, p40, and TTF-1. High-risk human papilloma virus (HR-HPV) was detected by in situ hybridization (ISH) using probes against E6 and E7 mRNA expressed in 18 different virus types. Vimentin, ER, and progesterone receptor were expressed in a significant minority of ECAs, mostly HPVAs, limiting their use in differential diagnosis of endometrioid carcinoma when unaccompanied by HPV-ISH or p16. HR-HPV ISH had superior sensitivity, specificity, and negative and positive predictive values compared with p16, as published previously. HNF-1beta did not have the anticipated discriminatory power for clear cell carcinoma, nor did MUC6 or CA-IX for gastric-type carcinoma. HNF-1beta and napsin A were variably expressed in clear cell carcinoma, with HNF-1beta demonstrating less specificity, as it was ubiquitously expressed in gastric-type carcinoma and in the majority of HPV-associated mucinous (predominantly intestinal-type and invasive ECA resembling stratified mucin-producing intraepithelial lesion [iSMILE]) and usual-type carcinomas. HIK1083 was expressed in nearly half of gastric-type carcinomas, but not in the vast majority of other subtypes. GATA3 was positive in 10% of usual-type adenocarcinomas and in single examples of other subtypes. Rare gastric-type and HPVA mucinous carcinomas displayed HER2 overexpression. Androgen receptor was positive in 6% of usual-type adenocarcinomas. Aberrant p53 expression was found in only 3.6% of usual-type HPVA carcinomas, but it was more prevalent in mucinous (intestinal type and iSMILE) HPVAs and non-human papilloma virus-associates (particularly in gastric-type carcinoma, >50% of cases). The following diagnostic classification algorithms were developed with the above data. Carcinomas without overt cytoplasmic mucin (endometrioid, usual-type endocervical, clear cell, and mesonephric carcinomas) can be subclassified using HR-HPV ISH, ER, and GATA3, whereas carcinomas with easily appreciated cytoplasmic mucin (endometrioid carcinoma with mucinous features, HPVA mucinous, and gastric-type carcinomas) can be subclassified with HR-HPV ISH and ER.

Although 2014 World Health Organization criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma, in practice, adenosquamous carcinoma diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphological, and clinical features and outcomes associated with adenosquamous carcinomas, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed adenosquamous carcinomas (including glassy cell carcinoma and related lesions) to confirm an adenosquamous carcinoma diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as adenosquamous carcinomas, 34 retained their adenosquamous carcinoma diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or adenosquamous carcinomas), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy cell carcinomas were reclassified as poorly differentiated usual-type carcinomas based on morphology and immunophenotype. There were significant immunophenotypic differences between adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas with regard to HPV (p < 0.0001), PAX8 (p = 0.038; more in adenosquamous carcinoma), p40 (p < 0.0001; more in adenosquamous carcinoma), p63 (p = 0.0018; more in adenosquamous carcinoma) and MUC6 (p  < 0.0001; less in adenosquamous carcinoma), HNF-1beta (p = 0.0023), vimentin (p = 0.0003), p53 (p = 0.0004), and CK7 (p = 0.0002) expression. Survival outcomes were similar between all groups. Adenosquamous carcinomas should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma, and categorizing them as such should be reconsidered.

Abstract

Background

Laryngeal squamous cell carcinoma (LSCC) is strongly associated with tobacco use, but recent reports suggest an increasing incidence of LSCC in patients without traditional risk factors, suggesting an alternative etiology of tumorigenesis. The purpose of this study is to characterize this non-smoking population and to compare immunohistochemical markers in tumor specimens from non-smokers and smokers with LSCC.

Methods

A retrospective chart review of patients with LSCC at Johns Hopkins Hospital (JHH) was performed. A tissue microarray (TMA) was constructed with tumor specimen from non-smokers with stage and age-matched smokers and stained for a variety of immunologic and molecular targets.

Results

In the JHH cohort of 521 patients, 12% (n = 63) were non-smokers. Non-smokers were more likely to be <45 years old at time of diagnosis (OR 4.13, p = 0.001) and to have glottic tumors (OR 2.46, p = 0.003). The TMA was comprised of tumors from 34 patients (14 non-smokers, 20 smokers). Only 2 patients (6%) were human-papillomavirus (HPV) positive by high-risk RNA in situ hybridization (ISH). There was no correlation between smoking status and p16 (p = 0.36), HPV-ISH positivity (p = 0.79), phosphatase and tensin homolog (PTEN, p = 0.91), p53 (p = 0.14), or programmed death-ligand 1 (PD-L1, p = 0.27) expression.

Conclusions

Non-smokers with LSCC are more likely to be younger at the time of diagnosis and have glottic tumors than smokers with LSCC. In TMA analysis of stage and age-matched specimens from smoker and non-smokers with LSCC, the pattern of expression for common molecular and immunologic markers is similar. Further, HPV does not appear to be a major causative etiology of LSCC in either smokers or non-smokers in our cohort of patients.

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells, but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinallamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium, and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced, whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens.