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Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5–52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

Topological data analysis (TDA) is a powerful method for reducing data dimensionality, mining underlying data relationships, and intuitively representing the data structure. The Mapper algorithm is one such tool that projects high-dimensional data to 1-dimensional space by using a filter function that is subsequently used to reconstruct the data topology relationships. However, domain context information and prior knowledge have not been considered in current TDA modeling frameworks. Here, we report the development and evaluation of a semi-supervised topological analysis (STA) framework that incorporates discrete or continuously labeled data points and selects the most relevant filter functions accordingly. We validate the proposed STA framework with simulation data and then apply it to samples from Genotype-Tissue Expression data and ovarian cancer transcriptome datasets. The graphs generated by STA for these 2 datasets, based on gene expression profiles, are consistent with prior knowledge, thereby supporting the effectiveness of the proposed framework.

Background Next-generation sequencing provides comprehensive information about individuals’ genetic makeup and is commonplace in precision oncology practice. Due to the heterogeneity of individual patient’s disease conditions and treatment journeys, not all targeted therapies were initiated despite actionable mutations. To better understand and support the clinical decision-making process in precision oncology, there is a need to examine real-world associations between patients’ genetic information and treatment choices. Methods To fill the gap of insufficient use of real-world data (RWD) in electronic health records (EHRs), we generated a single Resource Description Framework (RDF) resource, called PO2RDF (precision oncology to RDF), by integrating information regarding genes, variants, diseases, and drugs from genetic reports and EHRs. Results There are a total 2,309,014 triples contained in the PO2RDF. Among them, 32,815 triples are related to Gene, 34,695 triples are related to Variant, 8,787 triples are related to Disease, 26,154 triples are related to Drug. We performed two use case analyses to demonstrate the usability of the PO2RDF: (1) we examined real-world associations between EGFR mutations and targeted therapies to confirm existing knowledge and detect off-label use. (2) We examined differences in prognosis for lung cancer patients with/without TP53 mutations. Conclusions In conclusion, our work proposed to use RDF to organize and distribute clinical RWD that is otherwise inaccessible externally. Our work serves as a pilot study that will lead to new clinical applications and could ultimately stimulate progress in the field of precision oncology.

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.

ObjectivesSteatohepatitic hepatocellular carcinoma is a distinct variant of hepatocellular carcinoma strongly associated with underlying nonalcoholic steatohepatitis. The molecular biology of steatohepatitic hepatocellular carcinoma is not fully elucidated, and thus we aimed to investigate the molecular underpinnings of this entity.MethodsTranscriptomic analysis using RNAseq was performed on eight tumor-nonneoplastic pairs of steatohepatitic hepatocellular carcinoma with comparison to conventional hepatocellular carcinoma transcriptomes curated in The Cancer Genome Atlas. Immunohistochemistry was used to validate key RNA-level findings.ResultsSteatohepatitic hepatocellular carcinoma demonstrated a distinctive differential gene expression profile compared with The Cancer Genome Atlas curated conventional hepatocellular carcinomas (n = 360 cases), indicating the distinctive steatohepatitic hepatocellular carcinoma morphology is associated with a unique gene expression profile. Pathway analysis comparing tumor-nonneoplastic pairs revealed significant upregulation of the hedgehog pathway based on GLI1 overexpression and significant downregulation of carnitine palmitoyltransferase 2 transcript. Glutamine synthetase transcript was significantly upregulated, and fatty acid binding protein 1 transcript was significantly downregulated and immunohistochemically confirmed, indicating steatohepatitic hepatocellular carcinoma tumor cells display a zone 3 phenotype.ConclusionsSteatohepatitic hepatocellular carcinoma demonstrates a distinctive morphology and gene expression profile, phenotype of zone 3 hepatocytes, and activation of the hedgehog pathway and repression of carnitine palmitoyltransferase 2, which may be important in tumorigenesis.

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.

As patient derived xenograft (PDX) models are increasingly used for preclinical drug development, strategies to account for the nonhuman component of PDX RNA expression data are critical to its interpretation. A bioinformatics pipeline to separate donor tumor and mouse stroma transcriptome profiles was devised and tested. To examine the molecular fidelity of PDX versus donor tumors, we compared mRNA differences between paired PDX-donor tumors from nine ovarian cancer patients. 1,935 differentially expressed genes were identified between PDX and donor tumors. Over 90% (n = 1767) of these genes were down-regulated in PDX models and enriched in stroma-specific functions. Several protein kinases were also differentially expressed in PDX tumors, e.g. PDGFRA, PDGFRB and CSF1R. Upon in silico removal of these PDX-donor tumor differentially expressed genes, a stronger transcriptional resemblance between PDX-donor tumor pairs was seen (average correlation coefficient increases from 0.91 to 0.95). We devised and validated an effective bioinformatics strategy to separate mouse stroma expression from human tumor expression for PDX RNAseq. In addition, we showed most of the PDX-donor differentially expressed genes were implicated in stromal components. The molecular similarities and differences between PDX and donor tumors have implications in future therapeutic trial designs and treatment response evaluations using PDX models.

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.

Gene fusions involving the anaplastic lymphoma kinase ( ALK) gene occurs in approximately 3-5% of lung adenocarcinomas. The most common fusion partner is EML4 but several other fusion partners to the ALK kinase domain have been identified. Herein we report a lung adenocarcinoma case harboring a fusion between ALK and Striatin ( STRN ), with extended responses to targeted therapy. Using CT-guided needle biopsy approach, we successfully generated a patient-derived xenograft model from this tumor and molecularly characterized the STRN-ALK gene fusion.

Background: Differences in ER+ and ER- breast cancer tumor biology are well-documented, but little is known about the field of background benign breast in which those cancers arise. We evaluated the transcriptome of benign breast tissues from women with concurrent ipsilateral ER+ and ER- breast cancers (BC) to characterize coding and non-coding RNA profiles. This pilot study provides insight into the transcriptomic landscapes of benign breast tissues in patients with BC, and the microenvironment of the at-risk benign tissue. Methods: With institutional approval, cryobanked breast tissues from patients with concurrent ipsilateral ER+ BC (benign ER+BC, N=14) or ER- BC (benign ER-BC, N=10), were selected for benign tissues with similar epithelial:stromal ratios and grouped into pre (preM) and post-menopausal (PM) groups. Following RNA sequencing (Illumina TruSeq Stranded mRNA kit & Illumina HiSeq 4000), reads were processed (MAP-RSeq v3.0.0) and aligned (STAR aligner; hg38). Differential expression (DE) analysis (edgeR 2.6.2) identified DE genes from normalized RPKM reads (absolute log2 fold change (FC) > 1 and false discovery rate (FDR) < 0.10), corrected for intra-group biases. Over-representation analysis [Ingenuity pathway analysis (IPA), Ingenuity® Systems] and gene set enrichment analysis [(GSEA), GeneTrail 2.0] identified significantly-enriched pathways. Results: In the PM group, there were 144 DE transcripts between benign ER+BC and benign ER-BC, including coding RNAs (40%), antisense RNAs (35%) and lncRNAs (7%). In contrast, the preM group had no significantly DE genes between benign ER+BC and benign ER-BC. In the PM DE coding gene set, the top DE transcripts in benign ER+BC included many genes implicated in BC development# or ER+ BC progression (*) (e.g. up-regulated: KAAG1*, DNAJB7/HSP40*, TMEM151B, ZBTB32# p < 0.001 ; down-regulated: CPB1*, FOS, TPPP3#, CLEC3B#p < 0.001). Top canonical pathways altered in benign ER+BC included MAPK, PI3K, and acute phase response pathways (p 0.4) functionally grouped as immune function-related (T cell function and antigen presentation). Depleted pathways with NES > 0.4, (N=6) functionally grouped into proteasome-related, fatty acid biosynthesis and mitochondrial energy metabolism. Among the non-coding DE gene set, notably, the entire DE antisense RNA gene set (N=51 transcripts) was up-regulated in benign ER+ BC compared to benign ER-BC (P< 0.001) with a subset (N=11) showing marked up-regulation (> 4 log2FC). Among the DE antisense RNAs, 70% have reported roles in carcinogenesis or BC progression (e.g. KRT7-AS, NAV2-AS2, CCDC144NL-AS1, RP11-66B24.4, HSA-MIR4454). Conclusion: The benign breast transcriptome differs between postmenopausal women with ER+ vs ER- BC, with distinctive coding and non-coding RNA signatures. In postmenopausal women with ER+ BC, benign breast expresses a unique antisense RNA set and is enriched in genes implicated in BC development or progression. These data provide insight into at-risk benign breast and facilitate identification of potential biomarkers of carcinogenesis. Citation Format: Carter JM, Nair AA, Davila JI, Heinzen EP, Hoskin TL, Winham SJ, Radisky DC, Visscher DW, Degnim AC. A unique coding and non-coding benign breast transcriptome in post-menopausal ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-10.

Understanding the molecular events controlling melanoma progression is of paramount importance for the development of alternative treatment options for this devastating disease. Here we report a mechanism regulated by the oncogenic SOX2-GLI1 transcriptional complex driving melanoma invasion through the induction of the sialyltransferase ST3GAL1. Using in vitro and in vivo studies, we demonstrate that ST3GAL1 drives melanoma metastasis. Silencing of this enzyme suppresses melanoma invasion and significantly reduces the ability of aggressive melanoma cells to enter the blood stream, colonize distal organs, seed and survive in the metastatic environment. Analysis of glycosylated proteins reveals that the receptor tyrosine kinase AXL is a major effector of ST3GAL1 pro-invasive function. ST3GAL1 induces AXL dimerization and activation that, in turn, promotes melanoma invasion. Our data support a key role of the ST3GAL1-AXL axis as driver of melanoma metastasis, and highlight the therapeutic potential of targeting this axis to treat metastatic melanoma., Understanding the molecular events controlling melanoma progression are necessary to find improved therapeutics. Here, the authors report oncogenic SOX2-GLI1 transcriptional complex to drive melanoma invasion through the induction of the sialyltransferase ST3GAL1, and report ST3GAL1-AXL axis as driver of melanoma metastasis.

Objectives Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. Methods To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). Results Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. Conclusions Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.

Objectives Mucinous cystic neoplasm of the liver is characterized by neoplastic mucinous and/or biliary epithelium surrounded by ovarian-type stroma. Immunohistochemical studies have shown that the ovarian-type stroma expresses estrogen receptor, suggesting potential hormonal responsiveness. The molecular biology of mucinous cystic neoplasm of the liver remains poorly studied. Methods Transcriptome sequencing and immunohistochemistry were performed on a series of mucinous cystic neoplasms. Results Mucinous cystic neoplasm of the liver exhibited significantly increased RNA expression of ovarian stromal markers WT1, PR, and ER2 and sex cord stromal markers SF-1, inhibin-α, and calretinin compared with nonneoplastic liver. Immunohistochemistry confirmed the RNA-level data. Evidence for sex hormone biosynthesis was identified by significant overexpression of multiple estrogen biosynthetic enzymes. Expression of 17β-hydroxysteroid dehydrogenase 1 was confirmed immunohistochemically. Pathway analysis also identified significant upregulation of the hedgehog and Wnt pathways and significant downregulation of T-helper 1 and T-helper 2 pathways. Conclusions Mucinous cystic neoplasm of the liver recapitulates ovarian stroma at the morphologic, DNA, RNA, and protein levels. These data support the concept that this tumor likely arises from ectopic primitive gonadal tissue and/or stromal cells with capacity to transdifferentiate to ovarian cortical cells.

Background Traditionally, mutational burden and mutational signatures have been assessed by tumor-normal pair DNA sequencing. The requirement of having both normal and tumor samples is not always feasible from a clinical perspective, and led us to investigate the efficacy of using RNA sequencing of only the tumor sample to determine the mutational burden and signatures, and subsequently molecular cause of the cancer. The potential advantages include reducing the cost of testing, and simultaneously providing information on the gene expression profile and gene fusions present in the tumor. Results In this study, we devised supervised and unsupervised learning methods to determine mutational signatures from tumor RNA-seq data. As applications, we applied the methods to a training set of 587 TCGA uterine cancer RNA-seq samples, and examined in an independent testing set of 521 TCGA colorectal cancer RNA-seq samples. Both diseases are known associated with microsatellite instable high (MSI-H) and driver defects in DNA polymerase ɛ (POLɛ). From RNA-seq called variants, we found majority (> 95%) are likely germline variants, leading to C > T enriched germline variants (dbSNP) widely applicable in tumor and normal RNA-seq samples. We found significant associations between RNA-derived mutational burdens and MSI/POLɛ status, and insignificant relationship between RNA-seq total coverage and derived mutational burdens. Additionally we found that over 80% of variants could be explained by using the COSMIC mutational signature-5, -6 and -10, which are implicated in natural aging, MSI-H, and POLɛ, respectively. For classifying tumor type, within UCEC we achieved a recall of 0.56 and 0.78, and specificity of 0.66 and 0.99 for MSI and POLɛ respectively. By applying learnt RNA signatures from UCEC to COAD, we were able to improve our classification of both MSI and POLɛ. Conclusions Taken together, our work provides a novel method to detect RNA-seq derived mutational signatures with effective procedures to remove likely germline variants. It can leads to accurate classification of underlying driving mechanisms of DNA damage deficiency.

Multiple isodicentric Y chromosome [idic(Y)] is a cytogenetic abnormality consisting of duplicative copies of the short-arm of Y chromosome. Several mechanisms have been postulated to explain the f...

Aims Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harbouring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has been reported very recently. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma. Material and results A previously well 72-year-old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion. Conclusions Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/sclerosing rhabdomyosarcoma are discussed.

TO THE EDITOR: Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract (GI TLPD) is a newly recognized entity in the World Health Organization (WHO) classification of lymphoid neoplasms.[1][1] GI TLPD is defined as a clonal T-cell proliferation occurring in the GI tract, most

Aberrant expression of neuroendocrine markers is extremely rare in endothelial neoplasms, with only a single report describing three cases. Although originally classified as conventional angiosarcoma, further assessment of these tumors revealed a strikingly composite morphology composed of retiform and epithelioid elements reminiscent of composite hemangioendothelioma, a rare subtype of hemangioendothelioma. To further investigate these findings, available materials from 11 morphologically distinctive endothelial tumors showing neuroendocrine marker expression were retrieved from our archives. Immunohistochemistry for CD31, CD34, FLI-1, synaptophysin, chromogranin, D2-40, ERG, keratin (OSCAR), and CAMTA1 was performed. Total RNA from five cases were extracted and subjected to whole transcriptome sequencing. Clinical follow-up was obtained. These tumors were found to arise in five males and six females in patients from 9 to 55 years in age (median 47 years). They arose both in superficial (wrist, ankle, scalp, hip, and foot) and deep (periaortic tissues, C5 vertebra, pulmonary vein, and liver) locations. All contained elongated, retiform vascular channels lined by hyperchromatic 'hobnail' endothelial cells and a solid growth of uniform epithelioid cells reminiscent of epithelioid hemangioendothelioma. Hemangioma-like foci also lined by hobnail endothelial cells were frequently present. Mitotic activity was typically

Gastroblastoma is a rare distinctive biphasic tumor of the stomach. The molecular biology of gastroblastoma has not been studied, and no affirmative diagnostic markers have been developed. We retrieved two gastroblastomas from the consultation practices of the authors and performed transcriptome sequencing on formalin-fixed paraffin-embedded tissue. Recurrent predicted fusion genes were validated at genomic and RNA levels. The presence of the fusion gene was confirmed on two additional paraffin-embedded cases of gastroblastoma. Control cases of histologic mimics (biphasic synovial sarcoma, leiomyoma, leiomyosarcoma, desmoid-type fibromatosis, EWSR1-FLI1-positive Ewing sarcoma, Wilms' tumor, gastrointestinal stromal tumor, plexiform fibromyxoma, Sonic hedgehog-type medulloblastomas, and normal gastric mucosa and muscularis propria were also analyzed. The gastroblastomas affected two males and two females aged 9-56 years. Transcriptome sequencing identified recurrent somatic MALAT1-GLI1 fusion genes, which were predicted to retain the key domains of GLI1. The MALAT1-GLI1 fusion gene was validated by break-apart and dual-fusion FISH and RT-PCR. The additional two gastroblastomas were also positive for the MALAT1-GLI1 fusion gene. None of the other control cases harbored MALAT1-GLI1. Overexpression of GLI1 in the cases of gastroblastomas was confirmed at RNA and protein levels. Pathway analysis revealed activation of the Sonic hedgehog pathway in gastroblastoma and gene expression profiling showed that gastroblastomas grouped together and were most similar to Sonic hedgehog-type medulloblastomas. In summary, we have identified an oncogenic MALAT1-GLI1 fusion gene in all cases of gastroblastoma that may serve as a diagnostic biomarker. The fusion gene is predicted to encode a protein that includes the zinc finger domains of GLI1 and results in overexpression of GLI1 protein and activation of the Sonic hedgehog pathway.

// Nicholas B. Larson 1 , Shannon K. McDonnell 1 , Zach Fogarty 1 , Melissa C. Larson 1 , John Cheville 2 , Shaun Riska 1 , Saurabh Baheti 1 , Alexandra M. Weber 3 , Asha A. Nair 1 , Liang Wang 4 , Daniel O’Brien 1 , Jaime Davila 1 , Daniel J. Schaid 1 and Stephen N. Thibodeau 5 1 Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA 2 Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 3 Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA 4 Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA 5 Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Correspondence to: Nicholas B. Larson, email: Larson.nicholas@mayo.edu Keywords: prostate cancer, genetic risk, expression quantitative trait loci, mediation Received: April 26, 2017 Accepted: July 29, 2017 Published: September 08, 2017 ABSTRACT Large-scale genome-wide association studies have identified multiple single-nucleotide polymorphisms associated with risk of prostate cancer. Many of these genetic variants are presumed to be regulatory in nature; however, follow-up expression quantitative trait loci (eQTL) association studies have to-date been restricted largely to cis -acting associations due to study limitations. While trans -eQTL scans suffer from high testing dimensionality, recent evidence indicates most trans -eQTL associations are mediated by cis -regulated genes, such as transcription factors. Leveraging a data-driven gene co-expression network, we conducted a comprehensive cis -mediator analysis using RNA-Seq data from 471 normal prostate tissue samples to identify downstream regulatory associations of previously identified prostate cancer risk variants. We discovered multiple trans -eQTL associations that were significantly mediated by cis -regulated transcripts, four of which involved risk locus 17q12, proximal transcription factor HNF1B , and target trans -genes with known HNF response elements ( MIA2 , SRC , SEMA6A , KIF12 ). We additionally identified evidence of cis -acting down-regulation of MSMB via rs10993994 corresponding to reduced co-expression of NDRG1 . The majority of these cis -mediator relationships demonstrated trans -eQTL replicability in 87 prostate tissue samples from the Gene-Tissue Expression Project. These findings provide further biological context to known risk loci and outline new hypotheses for investigation into the etiology of prostate cancer.

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 Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit (“QA”), and QIAGEN GeneRead DNA FFPE Kit (“QGR”). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range ( T|G > A” mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency. Electronic supplementary material The online version of this article (10.1186/s12864-019-6056-8) contains supplementary material, which is available to authorized users.

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.

Introduction: Acquired resistance to platinum remains a major clinical problem in ovarian cancer since systemic therapies have limited efficacy in this setting. There is growing evidence that the tumor microenvironment (TME) contributes to such resistance, as well as the pathogenesis of the disease. The study of the TME is limited by technical challenges of separating the epithelial from the stromal component. To overcome this limitation, we performed RNA sequencing (RNA-Seq) for three pairs of sensitive and induced resistant patient-derived xenograft models since the epithelial transcripts originate from human tissue and the stroma component is murine. Methods: To determine which TME genes might be contributing to chemotherapy resistance, three platinum-sensitive ovarian cancer PDX lines were treated repeatedly with carboplatin/paclitaxel until resistance occurred. Primary platinum-sensitive and induced platinum resistant tumors were characterized histologically by H&E and Masson Trichrome staining. The three pairs of sensitive and resistant PDX models were then subjected to RNA-Seq. Raw sequencing data were processed through Xenome to classify the reads into epithelial carcinoma (human) or TME (mouse) through Mayo Clinic’s internal RNA-Seq pipeline for transcriptome quantification. Differential expression analysis between induced resistant and sensitive models was carried out for both carcinoma and TME components using edgeR paired analysis. Gene set enrichment analysis was used to identify overrepresented biologic functions for differentially expressed genes between the pairs. Top significantly varied, differentially expressed genes between the pairs were validated using species-specific qPCR. Results: Histologic analysis of the matched sensitive/resistant pairs revealed a marked increase of the stromal component in the resistant tumors evident by H&E and Masson trichrome staining. A total number of 95 and 312 genes were differentially expressed in the epithelial and TME component, respectively (FDR1.5). The stemness marker SOX2 was significantly upregulated in the human component of the resistant tumors as confirmed by qPCR. Genes involved in stroma remodeling were upregulated in the “murine” component of the resistant tumors, and upregulation was confirmed by qPCR (normalized to a murine housekeeper gene). Gene set enrichment analysis showed that the upregulated genes in TME of resistant models were enriched for cytoskeleton related functions, while the downregulated genes were enriched for extracellular matrix-related functions. Conclusion: A bioinformatics strategy able to map human and murine reads separately may identify resistance mechanisms attributed to the two tumor subcompartments and could lead to strategies to potentially target each separately or concurrently. Citation Format: Valentina Zanfagnin, Yuanhang Liu, Pritha Chanana, Xiaonan Pritha, Beverly Long, Joe Pathoulas, Brad Evans, Jaime Davila, Chen Wang, Saravut John Weroha. Transcriptional profiling of tumor stroma using ovarian cancer PDX models with induced platinum-resistance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B18.

Background: Despite a paucity of data on the benign breast transcriptome, reduction mammoplasty tissue is commonly used as a control for RNA biomarker discovery in breast cancer (BC). We evaluated the transcriptome of benign breast to characterize pre-menopausal and post-menopausal coding and non-coding RNA profiles. These profiles will provide insight into the metabolic landscape of benign breast and inform transcriptomic studies of BC and peri-tumoral microenvironments. Methods: With institutional approval, cryobanked fresh breast tissues from reduction mammoplasties (age 15-62y; follow-up: 4-10 y with no cancer events), with histologically-confirmed benign tissues with similar epithelial: stromal tissues were grouped into 4 age sets (< 30y, N=10; 30-39y, N=11; 40-49y, N=10; ≥50y (clinically post-menopausal), N=10). Following RNA extraction, library preparation (Illumina TruSeq Stranded mRNA kit), and sequencing (Illumina HiSeq 4000), reads were processed (MAP-RSeq v3.0.0) and aligned (STAR aligner; hg38). Differential expression (DE) analysis (edgeR 2.6.2) identified DE genes from normalized RPKM reads (absolute log2 fold change (FC) > 1 and false discovery rate (FDR) < 0.10), corrected for intra-group biases using medians (absolute FC cut-off of >1.5). Over-representation analysis [Ingenuity pathway analysis (IPA), Ingenuity® Systems] and gene set enrichment analysis [(GSEA), GeneTrail 2.0] identified significantly-enriched pathways. Results: Across the 4 age sets, 561 DE genes were identified. Compared to the post-menopausal (PM) set, the number of DE genes was highest in Conclusions: Pre and post-menopausal benign breast tissues have distinctive transcriptomic profiles. PM benign breast has down-regulated proliferation/cell-cycle-related pathways, and up-regulated genes in mitochondrial energy pathways, lipogenesis and inflammatory pathways. Notably, many of the top DE coding and non-coding RNAs in PM benign breast have been implicated in BC progression, highlighting their value in better understanding breast carcinogenesis and the need to characterize their functional roles in normal aging and menopause. Citation Format: Carter JM, Nair AA, Davila JI, Heinzen EP, Hoskin TL, Winham SJ, Radisky DC, Degnim AC, Visscher DW. Distinctive coding and non-coding RNA profiles of pre-menopausal and post-menopausal benign breast [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-06-01.

To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.

Purpose Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. Patients and Methods Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. Results Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. Conclusion Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion–positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration–approved and investigational targeted therapies.

RNA-seq is a mature and well-established method for studying the complexity of the transcriptome in the research setting. As this method moves from the research realm to the clinical context, new opportunities for the development of bioinformatics methods arise. During this talk I will present some of the challenges we have found during our work to release a clinical test for tumor samples using RNA-seq. During the first part of the talk I will focus on fusion detection, how it is affected by the degradation of the sample and how to quantify such effect using Fusion Sense [1]. I will also comment on the opportunities and challenges of annotating and predicting the clinical importance of fusions. During the second part of the talk I will comment on variant calling in RNA-seq and how to account for the effects of library preparation by using RVboost [2]. I will then show some preliminary work of leveraging this method in the context of estimating tumor mutational burden in Formalin-Fixed Paraffin-Embedded (FFPE) samples.

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced by an underlying plasma cell neoplasm. The clonotypic LC sequences are unique to each patient, and they cannot be reliably detected by either immunoassays or standard proteomic workflows that target the constant regions of LCs. We addressed this issue by developing a novel sequence template-based workflow to detect LC variable (LCV) region peptides directly from AL amyloid deposits. The workflow was implemented in a CAP/CLIA compliant clinical laboratory dedicated to proteomic subtyping of amyloid deposits extracted from either formalin-fixed paraffin-embedded tissues or subcutaneous fat aspirates. We evaluated the performance of the workflow on a validation cohort of 30 AL patients, whose amyloidogenic clone was identified using a novel proteogenomics method, and 30 controls. The recall and negative predictive values of the workflow, when identifying the gene family of the AL clone, were 93 and 98%, respectively. Application of the workflow on a clinical cohort of 500 AL amyloidosis samples highlighted a bias in the LCV gene families used by the AL clones. We also detected similarity between AL clones deposited in multiple organs of systemic AL patients. In summary, AL proteomic data sets are rich in LCV region peptides of potential clinical significance that are recoverable with advanced bioinformatics.

Background: Expansion of intronic (CTG·CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients. Results: RNA foci co-localizing with the splicing factor MBNL1 are found in FECD cells, and changes in mRNA splicing occur. Conclusion: Trinucleotide repeat expansion in FECD is associated with RNA focus formation and missplicing. Significance: RNA toxicity occurs in a disease affecting millions of patients., Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

Background and Aims Cholangiocytes are the target of a heterogeneous group of liver diseases, known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Methods Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a step-wise differentiation strategy toward iDCs using precise temporal exposure to key biliary morphogens and we characterized the cells using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Results Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, Western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in 3-dimensional culture. In vivo, the cells engraft within mouse liver following retrograde intra-biliary infusion. Conclusions In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modelling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.

Background : The 2016 revision to the World Health Organization classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In addition, CMML is characterized by a spectrum of cytogenetic and molecular abnormalities (Patnaik Leukemia 2014). In the current study, we looked for correlations between gene expression profiles (GEP) and morphologic or molecular categories of previously untreated patients with CMML. Methods : 35 patients with CMML were included in the study. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for 28 myeloid-relevant genes (Patnaik Blood C. J.). RNA sequencing (Stranded Total RNA- Illumina, San Diego, CA), was performed on 9 normal BM controls, and 35 CMML samples (25 peripheral blood (PB) and 10 BM). The RNA-Seq data was analyzed using MAPRSeq v.2.0. Normalization and differential expression analysis was performed using edgeR 2.6.2. We selected genes that were differentially expressed with a False Discovery Rate (FDR) Results: Among the 35 study patients, 75% were males and median age was 71 years (range, 55-87). 21 (60%), 9 (25%) and 5 (15%) patients were classified as CMML-0, 1 and 2, respectively. 16 (45%) had a dysplastic while 19 (55%) had a proliferative phenotype. At a median follow-up of 16 months, 5 (14%) deaths and 4 (14%) leukemic transformations were documented. Mutational frequencies included; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. Abnormal cytogenetics (n=9, 35%) included; -Y 8%, trisomy 8 6%, monosomy 7 3%, del(13q) 3% and monosomal karyotype 3%. i) Differential GEP in PB samples: 25 PB CMML samples were analyzed. Unsupervised GEP demonstrated two unique clusters (figure one); cluster one was enriched with dysplastic CMML (90%) and cluster two with proliferative CMML (64%). Consistent with the difference in the distribution of morphologic categories, in comparison to cluster one, patients in cluster two had a higher white blood count (p=0.003), higher monocyte count (p=0.02) and higher risk prognostication by the Mayo Model (p=0.016); there was no significant difference between the two clusters in terms of age and gender distribution, hemoglobin level, neutrophil and platelet counts, PB or BM blast content, cytogenetic risk stratification, ASXL1, TET2, RAS, CBL and SETBP1 mutational status. Genes significantly upregulated (FDR ii) Differential GEP in BM samples: 10 BM CMML samples were analyzed along with 10 normal controls. Samples and controls clustered separately and within the CMML samples two clusters were identified (figure two). These clusters had equal representations of dysplastic (n=5) and proliferative (n=5) CMML sub-types. Additional samples are being processed. Conclusions: Differential gene expression profiling in patients with CMML identifies two unique clusters, clinically demarcated, at least in PB samples, by "proliferative" and "dysplastic" CMML subtypes. The proliferative cluster is enriched in genes involved in cell cycle regulation and DNA damage response; whereas the dysplastic cluster is enriched in T-cell receptor and STAT3 signalling. The prognostic and therapeutic impact of these gene clusters remains to be assessed in a larger group of informative patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Novartis: Research Funding; Celgene: Research Funding.