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Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability–high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch–repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10−9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

Epithelial marker expression and/or epithelial differentiation, as well as “anomalous” expression of keratins, are features of some soft tissue tumors. Recently, we have encountered an unusual mesenchymal tumor composed of bland, distinctly eosinophilic, keratin-positive epithelial cells, which were almost entirely obscured by xanthogranulomatous inflammation. Six cases were identified (5 F, 1 M; 16–62 years (median 21 years)) arising in soft tissue (n = 4) and bone (n = 2) and ranging in size from 2 to 7 cm. The tumors were generally circumscribed, with a fibrous capsule containing lymphoid aggregates, and consisted in large part of a sheet-like proliferation of foamy histiocytes, Touton-type and osteoclast-type giant cells, and chronic inflammatory cells. Closer inspection, however, disclosed a distinct population of uniform, cytologically bland mononuclear cells with brightly eosinophilic cytoplasm arranged singly and in small nests and cords. Overt squamous and/or glandular differentiation was absent. By immunohistochemistry, these cells were diffusely positive with the OSCAR and AE1/AE3 keratin antibodies, and focally positive for high-molecular weight keratins; endothelial and myoid markers were negative and SMARCB1 was retained. RNA-seq identified a PLEKHM1 variant of undetermined significance in one case, likely related to this patient’s underlying osteopetrosis. Follow-up to date has been benign. In summary, we have identified a novel tumor of soft tissue and bone with a predilection for young females, provisionally termed “xanthogranulomatous epithelial tumor”. These unusual lesions do not appear to arise from adnexa, or represent known keratin-positive soft tissue tumors, and the origin of their constituent epithelial cells is obscure. The natural history of this distinctive lesion appears indolent, although study of additional cases and longer term follow-up are needed.

Aims: Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALKATI mutated melanoma as well as correlation with immunohistochemical (IHC) methods has not yet been described. Methods and results: Clinicopathologic characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in situ hybridization, and RNA based digital molecular analysis (NanoString Technologies) assays were performed on archival tissue from 173 stage III and 192 stage IV tumors. ALKATI was identified in 12.7% and 4.8% stage III and IV tumors, respectively. Discrete presentations of the ALKATI are seen: isolated ALKATI (N=20) and mixed ALKATI (combined ALKATI and ALKWT ; N=7). Isolated ALKWT expression (N=4) was seen with no ALK fusions. Stage III patients showed improved survival with ALKATI expression as compared to those with ALKWT or no expression [5-year survival 80% (95% CI: 57%-100%) versus 43% (95% CI: 34%-55%), p=0.013]. Clinicopathologic characteristics were not statistically significant. Strong diffuse cytoplasmic staining of ALK IHC (N=12), has a sensitivity of 52.2%, specificity 100%, PPV of 100% and NPV of 92.5% of detecting isolated ALKATI . Conclusion: Presence of ALKATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALKATI for targeted therapy.

Summary Uterine inflammatory myofibroblastic tumors (IMTs) have been reported in association with pregnancy and, in some instances, secondarily involve the placenta. The clinicopathological spectrum of these tumors in the setting of pregnancy is not well defined. We investigated the clinical, morphologic, immunohistochemical, molecular cytogenetic, and genetic features of 6 uterine IMTs occurring in pregnant women. Each tumor was discovered at parturition, and none was identified by prenatal ultrasound. Patient age ranged from 25 to 41 years (mean 31.5). Tumor size ranged from 1.5 to 9 cm (mean 4.7). Four of 6 had usual IMT features, with at least focal deciduoid change in 3. Necrosis was identified in 3 tumors; and multinucleated cells, in 3 tumors. Sex hormone receptor expression was consistent with estrogen receptor negative or focally weakly positive and progesterone receptor diffusely moderately or moderately to strongly positive in all 6 tumors. ALK immunohistochemistry was strongly positive in 5 tumors, and all of these had an ALK rearrangement detected by break-apart fluorescence in situ hybridization. Subsequent RNA sequencing of these 5 tumors identified a TIMP3-ALK fusion in 4 and a THBS1-ALK in 1. In the ALK-negative tumor, RNA sequencing detected a novel TIMP3-RET fusion that was confirmed by RET break-apart fluorescence in situ hybridization. Follow-up was available for 2 of 6 patients 5 and 19 months after diagnosis. Neither patient developed recurrence. ALK immunohistochemistry will distinguish most uterine IMTs, but if ALK expression and gene studies are negative, in the appropriate morphologic context, evaluation of other tyrosine kinase genes known to be more commonly altered in extrauterine IMTs such as ROS1, NTRK3, PDGFRβ, and RET may be necessary for diagnostic confirmation.

MAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%–27% variant allele frequency) and 1 showed a TPM3-NTRK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19–139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.

Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full-length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell-rich primary bone tumors. In this report, we present a case of a 16-year-old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X-USP6 promoter swap fusion. This result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin-specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate-specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.

Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKClinicopathological characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in-situ hybridisation and RNA-based digital molecular analysis assays were performed on archival tissue from 173 stage III and 192 stage IV tumours. ALKPresence of ALK

Background & objectives Biomarkers are increasingly required to molecularly characterize pancreatic ductal adenocarcinoma (PDAC) subgroup populations, to determine who may benefit from immune based targeted therapy. We evaluated the feasibility of gene expression signature detection and the respective landscape of specific tumor infiltrating lymphocytes (TILs), cancer/testis (CT) antigens, and immune checkpoints for possible future personalized immunotherapy eligibility. Methods Dedicated digital mRNA oncologic immune profiling of 770 genes using a Nanostring nCounter® PanCancer Immune Profiling Panel was performed using archived endoscopic ultrasound fine needle biopsy (EUS FNB) PDAC specimens as a case series in a tertiary care setting. Results The spectrum of mRNA gene expression within the tumor specimens revealed that 44.8%, 10.0% and 50.7% of evaluated genes had a ≥ 2-fold increase, a ≤ 2-fold reduction or between 2 change of mRNA expression, when compared to normal controls. The corresponding landscape of TILs, CT antigens, and immune checkpoints highlighted several possibilities that could potentially be amenable to targeted personalized immunotherapy. This includes members of the Tumor Associated Macrophage family (CD68, CXCL5, and MARCO), members of the CT antigen family (PRAME, TTK and PBK) and the “second generation” checkpoints TIM3 and BTLA. Conclusions Our study represents the ability to successfully perform digital mRNA expression profile analyses to immunophenotype PDAC EUS FNB specimens by evaluating the expression of >730 genes within the tumor immune microenvironment. This may facilitate the search for novel therapeutic targets, offering the opportunity to go beyond immune monotherapy, but perhaps to use combined immunomodulatory agents.

Formalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

Background Uterine leiomyomas, in contrast to sarcomas, tend to cease growth following menopause. In the setting of a rapidly enlarging uterine mass in a postmenopausal patient, clinical distinction of uterine leiomyoma from sarcoma is difficult and requires pathologic examination. Case presentation A 74-year-old woman presented with postmenopausal bleeding and acute blood loss requiring transfusion. She was found to have a rapidly enlarging uterine mass clinically suspicious for sarcoma. An abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. A 15.5 cm partially necrotic intramural mass was identified in the uterine corpus. The tumor was classified as a cellular leiomyoma. RNA sequencing identified a KAT6B-KANSL1 fusion that was confirmed by RT-PCR and Sanger sequencing. After 6 months of follow-up, the patient remains asymptomatic without evidence of disease. Conclusion Prior studies of uterine leiomyomas have identified KAT6B (previously MORF) rearrangements in uterine leiomyomas, but this case is the first to identify a KAT6B-KANSL1 gene fusion in a uterine leiomyoma. While alterations of MED12 and HMGA2 are most common in uterine leiomyomas, a range of other genetic pathways have been described. Our case contributes to the evolving molecular landscape of uterine leiomyomas.

System-on-a-chip technology allows a level of integration that can be leveraged to develop inexpensive high-performance, low-power computing nodes. When used in aggregate, this approach promises to challenge conventional supercomputer architectures in the high-performance computing arena. Systems under consideration reach into the hundreds of thousand nodes per machine. Architecture for these systems are described.