Faculty Bibliography

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.ajpath.2020.07.001. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

OBJECTIVE:To characterize the ecological effects of biologic therapies on the gut bacterial and fungal microbiome of psoriatic arthritis (PsA)/spondyloarthritis (SpA) patients. METHODS:Fecal samples from PsA/SpA patients pre- and post-treatment with tumor necrosis factor inhibitors (TNFi; n=15) or an anti-interleukin (IL)-17A monoclonal antibody inhibitor (IL-17i; n=14) underwent sequencing (16S, ITS and shotgun metagenomics) and computational microbiome analysis. Fecal levels of fatty acid metabolites and cytokines/proteins implicated in PsA/SpA pathogenesis or intestinal inflammation were correlated with sequence data. Additionally, ileal biopsies obtained from SpA patients who developed clinically overt Crohn's disease (CD) after treatment with IL-17i (n=5) were analyzed for expression of IL-23/Th-17 related cytokines, IL-25/IL-17E-producing cells and type-2 innate lymphoid cells (ILC2s). RESULTS:There were significant shifts in abundance of specific taxa after treatment with IL-17i compared to TNFi, particularly Clostridiales (p=0.016) and Candida albicans (p=0.041). These subclinical alterations correlated with changes in bacterial community co-occurrence, metabolic pathways, IL-23/Th17-related cytokines and various fatty acids. Ileal biopsies showed that clinically overt CD was associated with expansion of IL-25/IL-17E-producing tuft cells and ILC2s (p<0.05) compared to pre-IL-17i treatment levels. CONCLUSION/CONCLUSIONS:In a subgroup of SpA patients, the initiation of IL-17A blockade correlated with features of subclinical gut inflammation and intestinal dysbiosis of certain bacterial and fungal taxa, most notably C. albicans. Further, IL-17i-related CD was associated with overexpression of IL-25/IL-17E-producing tuft cells and ILC2s. These results may help to explain the potential link between inhibition of a specific IL-17 pathway and the (sub)clinical gut inflammation observed in SpA.

Background: Encapsulated papillary carcinomas (EPC) of the breast is a variant of papillary carcinoma that are confined to a cystic space, surrounded by a fibrous capsule and lack the myoepithelial coat. Despite the latter finding, it is recommended that EPC be staged as pTis due to its indolent course. Concurrent frank invasive carcinomas are staged commensurate with their size. We sought to investigate the molecular pathways differentially expressed in pure EPC and EPC with frank invasion at the genomic and transcriptomic level. In addition, we compared EPC with its corresponding invasive ductal carcinoma (IDC) at the transcriptomic level.
Design(s): We selected 3 cases of pure EPC (C1-C3) and 3 cases of EPC (C4e-C6e) with corresponding IDC (C4i-C6i).We performed whole transcriptome analysis on laser-capture microdissected samples from formalin-fixed, paraffin-embedded tissue. We used CloneTech Mammalian stranded pico kit for sequencing RNA. KEGG pathway analysis and Gene Ontology (GO) analysis was performed using the cluster Profiler R package (v3.0.0) and Database for Annotation, Visualization and Integrated Discovery. DNA analysis was performed using our in-house next generation sequencing hybrid capture covering 580 genes on C1-C3 and C4e-C6e.
Result(s): There were 5 female and 1 male patients. The mean age was 73 years (range 62-90). All cases were hormone receptor positive. C4e-C6e showed upregulation of NTRK2 and MAGI2 (lg2FC= 3.14 and lg2FC = 3.0 fold, respectively) and downregulation of PRKACB (lg2FC= -4.4) compared to C1-C3 on RNAseq. C4i-C6i showed upregulation of collagen-related genes (COL10A1, COL11A1, COL14A1, COL16A1, COL1A1, COL3A1, COL8A1) (lg2FC range: 6.28 fold change) and ADAM12/ADAMTS2 (lg2FC=6.2 and lg2FC= 6.9 fold change) compared to C4e-C6e (FDR =0.014). Pathway analysis showed upregulation of collagen fibril organization and extracellular matrix organization pathways in C4i-C6i compared to C4e-C6e and upregulation of kinase activity pathway (GO: 0016301) in C4e-C6e compared to C1-C3.Recurrent PIK3CA hotspot non-synonymous mutation was identified in C3, C4e, C5e and C6e (c.G1633A in C5 and c.A3140G in C3, C4 and C6).
Conclusion(s): Our findings suggest that kinase and matrix metalloproteinase pathways contribute to EPC with invasion compared to pure EPC cases. Furthermore, enrichment of collagen-related genes in IDCs compared to their corresponding EPC suggest a synergistic potential with the aforementioned pathways. Mechanistic studies are warranted to validate the findings

Plakophilin-2 (PKP2) is classically defined as a component of the desmosome. Besides its role in cell-cell adhesion, PKP2 can modulate transcription through intracellular signals initiated at the site of cell-cell contact. Mutations in PKP2 associate with arrhythmogenic right ventricular cardiomyopathy (ARVC). Recent data demonstrate that inflammation plays a key role in disease progression; other results show an abundance of anti-heart antibodies in patients with confirmed diagnosis of ARVC. Here, we test the hypothesis that, in adult cardiac myocytes, PKP2 transcript abundance is endogenously linked to the abundance of transcripts participating in the inflammatory/immune response. Cardiac-specific, tamoxifen (TAM)-activated PKP2-knockout mice (PKP2cKO) were crossed with a RiboTag line to allow characterization of the ribosome-resident transcriptome of cardiomyocytes after PKP2 knockdown. Data were combined with informatics analysis of human cardiac transcriptome using GTEx. Separately, the presence of non-myocyte cells at the time of analysis was assessed by imaging methods. We identified a large number of transcripts upregulated consequent to PKP2 deficiency in myocytes, inversely correlated with PKP2 abundance in human transcriptomes, and part of functional pathways associated with inflammatory/immune responses. Our data support the concept that PKP2 is transcriptionally linked, in cardiac myocytes, to genes coding for host-response molecules even in the absence of exogenous triggers. Targeted anti-inflammatory therapy may be effective in ARVC.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.

Multifocal breast cancer (MFBC), ductal type, has been hypothesized to arise by one of two mechanisms: either through intramammary/intralymphatic spread from a single index tumor (MBC-1), or as multiple independent tumors with each focus carrying its corresponding ductal carcinoma in-situ (MBC-2). In order to improve our understanding of MFBC pathogenesis, we employed laser capture microdissection coupled with whole-exome sequencing to study clonal origin in MFBC. We selected three cases of MBC-1 (C1 to C3) and MBC-2 (C4 to C6) and analyzed three foci from each case. MBC-1 cases were histologically similar and showed a strong predilection for satellite foci, vascular invasion and nodal metastasis when compared to MBC-2. Our bioinformatics approach provided strong evidence for clonal relationships in MBC-1, as demonstrated by distinct clusters of genes conserved across all tumor foci. Conversely, no gene clusters were shared across all the foci in MBC-2, suggesting multiple independent tumors. These findings provide further support for the two distinct pathogenetic mechanisms in MFBC.

BACKGROUND: While most hemangioblastomas (~70%) are sporadic and occur predominantly in the cerebellum, they may present as well as familial form associated with von Hippel-Lindau (VHL) syndrome, an autosomal dominant disorder caused by germline mutations of the VHL gene that trigger nuclear translocation of hypoxia-inducible factor (HIF)- 1alpha and angiogenesis. Although inactivation of VHL, a tumor suppressor gene, has been observed in hemangioblastomas, the underlying pathogenic mechanisms responsible for familial and sporadic hemangioblastomas remain incompletely understood.
METHOD(S): Whole exome sequencing of cerebellar hemangioblastoma tumors and matched blood leukocytes from 24 patients, age 24-63, was performed. After preparation and amplification of barcoded libraries, exomes were captured using Kapa Biosystems methodology and paired-end sequenced on Illumina HiSeq 2500 to an average 100-fold coverage. Following read alignment to hg19 genome, tumor and germline (leukocyte) sequences were compared, and pathogenic single nucleotide variants (SNVs) identified and validated by re-sequencing followed by pathway analysis. Additionally, tumor RNA isolated using Maxwell Promega was sequenced on Illumina instrument and the expression counts determined and normalized.
RESULT(S): We found 314 pathogenic and/or highly deleterious mutations (both germline and somatic) with a median of 13 mutations per patient. Five patients had VHL syndrome (germline VHL mutation) and 4 carried somatic VHL mutations. Among the VHL tumors, 82 mutations were identified, including HNF1B, NOTCH1 and TCF7L1, suggesting a potential contribution of altered RNA metabolism based upon pathway analysis. Among all hemangioblastomas, germline growth factor receptor variants (FGFR4 p.G388R (14/23 (61%) patients), IGF1R, PDGFRA and TYK2) known to activate STAT3 signaling and induce HIF-1alpha and angiogenesis, were identified. Non-hierarchical clustering of RNA sequencing data revealed two transcriptionally-distinct subtypes of hemangioblastomas.
CONCLUSION(S): Our findings indicate that hemangioblastomas can also occur by germline mutations known to activate STAT3 signaling, which may have significant implication in genetic testing and counseling of patients with hemangioblastomas

Recurrent somatic missense mutations in histone H3 genes have been identified in subsets of pediatric cancers. H3K36 histone mutations have recently been recognized as oncogenic drivers in rare subsets of malignant soft tissue sarcomas but have not been reported in histiocytic neoplasms. Currently, the histological and molecular spectrum, as well as the clinical behavior of H3K36-mutant soft tissue malignancies, is largely unknown. We describe a pediatric patient with a HIST1H3B K36I-mutant histiocytic tumor arising in the skull. After the failure of upfront therapy for histiocytosis and development of widely disseminated metastatic disease, the patient had an exceptional response to empiric chemotherapy and remains in complete disease remission for more than 5 years. Our report expands the histological spectrum of H3K36M/I-mutant soft tissue malignancies to histiocytic neoplasms and indicates that multiagent sarcoma-like chemotherapy can be highly effective even in the setting of widely disseminated metastatic disease.