Faculty Bibliography

Erdheim-Chester disease (ECD) is a rare clonal histiocytic process that is characterized by a foamy (xanthomatous) proliferation often associated with Touton giant cells. The diagnosis is often challenging and not exclusively a histologic diagnosis, as it requires correlation with unique clinical, radiographic and recently described molecular findings. Activating mutations involving the MAPK pathway including BRAF, ARAF, N/KRAS and MEK are recurrent in the disease. However, it is increasingly being described that mutations associated with clonal hematopoiesis are also found in bone marrow specimens of patients with Erdheim-Chester disease (ECD), as well as higher frequency of overt concomitant myeloid malignancy including acute myeloid leukemia, myeloproliferative neoplasms, myelodysplastic syndromes, and mixed myeloproliferative neoplasms/myelodysplastic syndromes. Herein, we report a unique case of a patient presenting with BRAF-V600E-positive ECD with with peripheral blood findings consistent with a concurrent myeloid malignancy featuring co-occurrence of NRAS and IDH-2 mutations.

BACKGROUND:A 64-year-old man presented with a 7.8 cm lipomatous thyroid mass discovered on magnetic resonance imaging. METHODS:After two non-diagnostic fine needle aspirations (FNAs) were performed, computed tomography (CT) revealed features concerning for malignancy including central necrosis and infiltrative borders. A third FNA was still non-diagnostic. Total thyroidectomy was performed. RESULTS:Upon pathologic examination, the final diagnosis was primary thyroid angiolipoma. The lesion contained central fat necrosis with ischemic features, attributable to the FNAs. CONCLUSION/CONCLUSIONS:Ours is the third published case report of this rare entity. To date, no lipomatous thyroid tumor has undergone extensive genomic testing. Next-generation sequencing of our case revealed multiple genetic alterations, supporting the concept of angiolipomas being true neoplasms. Whereas the two previously reported cases in the literature were radiographically much smaller and appeared indolent, the large tumor in our case exhibited radiographic features concerning for liposarcoma, which belied the benign final pathologic diagnosis. Our case demonstrates that conservative surgical management (partial thyroidectomy) may be considered for lipomatous thyroid tumors, with further interventions to be determined only after final pathologic diagnosis.

There is currently no targeted therapy to treat NF1-mutant melanomas. Herein, we compared the genomic and transcriptomic signatures of NF1-mutant and NF1-WT melanoma to reveal potential treatment targets for this subset of patients. Genomic alterations were verified using qPCR, and differentially expressed genes were independently validated using TCGA data, and immunohistochemistry (IHC). Digital spatial profiling (DSP) with multiplex IHC and immunofluorescence (IF) were used to validate the signatures. The efficacy of combinational regimens driven by these signatures was tested through in vitro assays using low-passage cell lines. Pathogenic NF1 mutations were identified in 27% cases. NF1-mutant melanoma expressed higher proliferative markers MK167 and CDC20 compared to NF1-WT (P=0.008), which was independently validated both in the TCGA dataset (P=0.01, P=0.03) and with IHC (P=0.013, P=0.036), respectively. DSP analysis showed upregulation of LY6E within the tumor cells [FDR<0.01, lg2FC>1], confirmed with multiplex IF showing co-localization of LY6E in melanoma cells. The combination of MEK and CDC20 co-inhibition induced both cytotoxic and cytostatic effects, decreasing CDC20 expression in multiple NF1-MUT cell lines. In conclusion, NF1-mutant melanoma is associated with a distinct genomic and transcriptomic profile. Our data support investigating CDC20 inhibition with MAPK pathway inhibitors as a targeted regimen in this melanoma subtype.

Introduction: Gene rearrangements play a critical role in the development of brain tumors. RNA next-generation sequencing (NGS) panels cover a limited number of genes, are rarely successful in FFPE samples > 5 years old, and cannot detect rearrangements between genes and non-coding regulatory regions. We evaluated whole genome Hi-C NGS for detection of gene fusions and cryptic rearrangements.
Method(s): DNA was extracted from FFPE scrolls of 55 glial and non-glial brain tumors and processed using Arima-HiC+ FFPE Sample protocol, consisting of chromatin fragmentation, labeling, and re-ligation, followed by DNA purification and library preparation for paired-end Illumina sequencing with an average of 10X genome coverage (100M PE reads per sample). Data were analyzed using the Arima-SV pipeline using Juicer and HiCUP, SV detection using HiC-Breakfinder, loop calling using Juicer Tools, and integrative data visualization using Juicebox. Overexpression of putative driver genes was confirmed by immunohistochemistry.
Result(s): Hi-C libraries were prepared and sequenced from FFPE tissues including samples that failed RNA NGS. Hi-C successfully detected gene-gene fusions including actionable EML4-NTRK3, ETV6-NTRK3, fusions. We detected rearrangements missed by RNA NGS (i.e., complex MYBL1 rearrangement) or between non-coding regions and known cancer genes (i.e. PDL1, PAX5, NRAS, TERT, KAT6A, GATA6, and ARID1B). Since Hi-C data captures 3D genome structural features such as chromatin loops and topological domains, datasets were of high quality and capable of detecting up to 13,000 chromatin loops per tumor.
Conclusion(s): Genome-wide Hi-C NGS is successful in detecting gene fusions and cryptic rearrangements between coding and non-coding regions in archival FFPE tissue including degraded samples. Because Hi-C data captures 3D genome structures, these datasets simultaneously inform gene regulatory mechanisms that may play a role in oncogenesis or tumor progression. Whole-genome Hi-C NGS expands our ability to detect actionable and novel drivers, and potentially new therapeutic targets in a single NGS workflow

Background: Digital papillary adenocarcinoma (DPAC) is a rare but aggressive cutaneous malignant sweat gland neoplasm that occurs on acral sites and mimics other benign entities leading to diagnostic dilemmas. We investigate genomic and transcriptomic signatures unique to DPAC that would help differentiate it from other benign entities and aim to unveil unique transcriptomic signatures inherent to its biology.
Design(s): 9 DPAC and 10 hidradenoma (HD) cases were selected (Table 1). DNA analysis used targeted 607 gene FDA validated panel (clinically validated). Customized RNA panel targeting 104 genes (FusionSeeqer) was used for fusion analysis. All pipelines used are clinically validated. Transcriptomic analysis used nCounter Pan Cancer IO 360TM panel (770 genes) with subsequent analysis using our own pipelines n R studio. Accurate transcript quantification (lg2FC) was performed after normalization to standard housekeeping genes. DESEQ2 was used for the gene level differential gene expression (DGE) analysis after normalization to reference group benign HDs (FDR<0.01, fold change: > 2 or < -2) with subsequent KEGG pathway analysis.
Result(s): DPAC cases showed very low tumor mutational burden on genomic analysis (range 0-1 mut/mb). Gene rearrangements were more frequent in HD compared to DPAC (4/4 vs 2/7, p= 0.03). Mastermind-like family of protein genes (MAML2) rearrangements were more frequent in HD, while DPAC showed novel zinc finger gene (PLAG1) rearrangements (TRPS1- PLAG1; n=1) (Fig 1A). Unsupervised clustering analysis and subsequent DGE analysis revealed 100 significantly differentially expressed genes between DPAC and HD. MAGEA4, IL2, IFNG, and COL11A2 showed a significant upregulation in DPAC (lg2FC =2.6, 2.93, 2.98, 3.04, FDR range = 0.0001 to 0.00004) respectively (Fig 1B). Pathway analysis identified enrichment of JAK/STAT pathway, which is triggered by the upregulated cytokines in DPAC (Fig 1C).
Conclusion(s): While morphologically overlapping, DPAC and HD are biologically distinct. MAGEA4 upregulation seen in DPAC could serve as potential marker for both diagnostic and therapeutic purposes, since it could be potentially targeted with adoptive Tcell therapy (ADP-A2M4). Validation of the findings on a larger cohort is underway

Background: There is paucity of transcriptomic data concerning primary cutaneous CD8+ lymphoproliferative disorders. Herein we aim to investigate the transcriptomic profile of CD8+ mycosis fungoides (CD8+ MF) and type D lymphomatoid papulosis (Type D LyP), in order to better characterize them at and assess whether the two entities are biologically related.
Design(s): Adequate RNA was successfully extracted from formalin-fixed paraffin embedded sections derived from 7 CD4+ MF, 8 CD8+ MF and 4 type D LyP. After passing quality control, whole transcriptome sequencing (WTS) was performed. Fusion detection was performed using star-fusion (version 1.10.0). Accurate transcript quantification from RNASEQ data was performed using RSEM (version 1.3.1). DESEQ2 was used for differential gene expression (DGE) analysis, comparing CD8+ MF vs CD4+MF, CD8+ MF vs type D LyP groups, after normalization against normal skin controls.
Result(s): Unsupervised clustering classified CD4+ MF and CD8+ MF into two distinct groups and identified 100 genes that were differentially expressed at a significant level between both entities (Figure 1 a). CD8+ MF shows a profound down regulation of skin-homing chemokine receptors, immune signaling-related molecules or transcription factors and upregulation of long non-coding RNAs (lncRNAs) and small nuclear RNAs (snRNAs) (Figure 1a). KEGG pathway analysis showed an upregulation of pathways related to linoleic acid metabolism, as well as retinoid acid, PPAR and adipocytokine signaling in CD8+ MF (Figure 1b). Unsupervised clustering did not segregate CD8+ MF and Type D LyP. Nevertheless, DGE analysis identified 546 genes that were differentially expressed between both (FDR<0.01, fold change: > 1 or < -1, Figure 1c). KEGG pathway analysis showed that Wnt signaling is significantly upregulated in CD8+ MF compared to Type D LyP. A recurrent mRNA-lncRNA fusion secondary to interstitial deletion involving RP11-367G6.3-FAM65B (Figure 1d) was identified in 5 cases from 4 unique patients, including 2 type D LyP and 2 CD8+ MF patients.
Conclusion(s): WTS shows that CD8+ MF has unique signatures compared to CD4+MF pertaining to lncRNAs and snRNAs. Type D LyP and CD8+ MF display similar genomic rearrangements, yet pathway analysis highlights different genes contributing to their pathogenesis