Deep neural network based tissue deconvolution of circulating tumor cell RNA.

Prior research has shown that the deconvolution of cell-free RNA can uncover the tissue origin. The conventional deconvolution approaches rely on constructing a reference tissue-specific gene panel, which cannot capture the inherent variation present in actual data. To address this, we have developed a novel method that utilizes a neural network framework to leverage the entire training dataset. Our approach involved training a model that incorporated 15 distinct tissue types. Through one semi-independent and two complete independent validations, including deconvolution using a semi in silico dataset, deconvolution with a custom normal tissue mixture RNA-seq data, and deconvolution of longitudinal circulating tumor cell RNA-seq (ctcRNA) data from a cancer patient with metastatic tumors, we demonstrate the efficacy and advantages of the deep-learning approach which were exerted by effectively capturing the inherent variability present in the dataset, thus leading to enhanced accuracy. Sensitivity analyses reveal that neural network models are less susceptible to the presence of missing data, making them more suitable for real-world applications. Moreover, by leveraging the concept of organotropism, we applied our approach to trace the migration of circulating tumor cell-derived RNA (ctcRNA) in a cancer patient with metastatic tumors, thereby highlighting the potential clinical significance of early detection of cancer metastasis.

The inflammatory response of human pancreatic cancer samples compared to normal controls.

Pancreatic ductal adenocarcinoma (PDAC) is a poor prognosis cancer with an aggressive growth profile that is often diagnosed at late stage and that has few curative or therapeutic options. PDAC growth has been linked to alterations in the pancreas microbiome, which could include the presence of the fungus Malassezia. We used RNA-sequencing to compare 14 matched tumor and normal (tumor adjacent) pancreatic cancer samples and found Malassezia RNA in both the PDAC and normal tissues. Although the presence of Malassezia was not correlated with tumor growth, a set of immune- and inflammatory-related genes were up-regulated in the PDAC compared to the normal samples, suggesting that they are involved in tumor progression. Gene set enrichment analysis suggests that activation of the complement cascade pathway and inflammation could be involved in pro PDAC growth.

Humanized Patient-derived Xenograft Models of Disseminated Ovarian Cancer Recapitulate Key Aspects of the Tumor Immune Environment within the Peritoneal Cavity.

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood-derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials. Significance: huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Dominant Gene Expression Profiles Define Adenoid Cystic Carcinoma (ACC) from Different Tissues: Validation of a Gene Signature Classifier for Poor Survival in Salivary Gland ACC.

Adenoid cystic carcinoma (ACC) is an aggressive malignancy that most often arises in salivary or lacrimal glands but can also occur in other tissues. We used optimized RNA-sequencing to analyze the transcriptomes of 113 ACC tumor samples from salivary gland, lacrimal gland, breast or skin. ACC tumors from different organs displayed remarkedly similar transcription profiles, and most harbored translocations in the MYB or MYBL1 genes, which encode oncogenic transcription factors that may induce dramatic genetic and epigenetic changes leading to a dominant 'ACC phenotype'. Further analysis of the 56 salivary gland ACC tumors led to the identification of three distinct groups of patients, based on gene expression profiles, including one group with worse survival. We tested whether this new cohort could be used to validate a biomarker developed previously with a different set of 68 ACC tumor samples. Indeed, a 49-gene classifier developed with the earlier cohort correctly identified 98% of the poor survival patients from the new set, and a 14-gene classifier was almost as accurate. These validated biomarkers form a platform to identify and stratify high-risk ACC patients into clinical trials of targeted therapies for sustained clinical response.

Genomic landscape of lymphatic malformations: a case series and response to the PI3Kα inhibitor alpelisib in an N-of-1 clinical trial.

Background: Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases. Methods: We examined the genomic landscape of a patient cohort of LMs (n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel. Results: These LMs had low mutational burden with hotspot PIK3CA mutations (n = 20) and NRAS (n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status. Conclusions: Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations. Funding: R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953. Clinical trial number: NCT03941782.

Two-step mixed model approach to analyzing differential alternative RNA splicing.

Changes in gene expression can correlate with poor disease outcomes in two ways: through changes in relative transcript levels or through alternative RNA splicing leading to changes in relative abundance of individual transcript isoforms. The objective of this research is to develop new statistical methods in detecting and analyzing both differentially expressed and spliced isoforms, which appropriately account for the dependence between isoforms and multiple testing corrections for the multi-dimensional structure of at both the gene- and isoform- level. We developed a linear mixed effects model-based approach for analyzing the complex alternative RNA splicing regulation patterns detected by whole-transcriptome RNA-sequencing technologies. This approach thoroughly characterizes and differentiates three types of genes related to alternative RNA splicing events with distinct differential expression/splicing patterns. We applied the concept of appropriately controlling for the gene-level overall false discovery rate (OFDR) in this multi-dimensional alternative RNA splicing analysis utilizing a two-step hierarchical hypothesis testing framework. In the initial screening test we identify genes that have differentially expressed or spliced isoforms; in the subsequent confirmatory testing stage we examine only the isoforms for genes that have passed the screening tests. Comparisons with other methods through application to a whole transcriptome RNA-Seq study of adenoid cystic carcinoma and extensive simulation studies have demonstrated the advantages and improved performances of our method. Our proposed method appropriately controls the gene-level OFDR, maintains statistical power, and is flexible to incorporate advanced experimental designs.

Oncogenic Orphan Nuclear Receptor NR4A3 Interacts and Cooperates with MYB in Acinic Cell Carcinoma.

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4;9), t(9;12), t(8;9) or t(2;4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

N-Terminal Truncated Myb with New Transcriptional Activity Produced Through Use of an Alternative MYB Promoter in Salivary Gland Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland tumor that frequently displays perineural invasion and is often associated with translocations or overexpression of the MYB oncogene. Detailed analyses of MYB transcripts from ACC patient samples revealed that ACC tumors utilize an alternative MYB promoter, which is rarely used in normal cells or other tumor types. The alternative promoter transcripts produce N-terminally truncated Myb proteins lacking a highly conserved and phosphorylated domain, which includes the pS11 epitope that is frequently used to detect Myb proteins. In RNA-seq assays, Myb isoforms lacking the N-terminal domain displayed unique transcriptional activities, regulating many genes differently than full-length Myb. Thus, a regulatory pathway unique to ACC activates the alternative MYB promoter, leading to the production of a truncated Myb protein with altered transcriptional activities. This could provide new therapeutic opportunities for ACC patients.

Tumor Heterogeneity as a Predictor of Response to Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) is the standard of care for locally advanced adenocarcinoma of the rectum, but it is currently unknown which patients have disease that will respond. This study tested the correlation between response to nCRT and intratumoral heterogeneity using next-generation sequencing assays. PATIENTS AND METHODS: DNA was extracted from formalin-fixed, paraffin-embedded biopsy samples from a cohort of patients with locally advanced rectal adenocarcinoma (T3/4 or N1/2 disease) who received nCRT. High read-depth sequencing of > 400 cancer-relevant genes was performed. Tumor mutations and variant allele frequencies were used to calculate mutant-allele tumor heterogeneity (MATH) scores as measures of intratumoral heterogeneity. Response to nCRT was pathologically scored after surgical resection. RESULTS: Biopsy samples from 21 patient tumors were analyzed. Eight patients had disease noted to have complete response, 2 moderate, 4 minimal, and 7 poor. Higher MATH scores correlated with poorer response to treatment, demonstrating significantly increased tumor heterogeneity compared to complete response (P = .039). CONCLUSION: The application of MATH scores as a measure of tumor heterogeneity may provide a useful biomarker for treatment response in locally advanced rectal cancer.

Epigenetic silencing of SOCS5 potentiates JAK-STAT signaling and progression of T-cell acute lymphoblastic leukemia.

Activating mutations in cytokine receptors and transcriptional regulators govern aberrant signal transduction in T-cell lineage acute lymphoblastic leukemia (T-ALL). However, the roles played by suppressors of cytokine signaling remain incompletely understood. We examined the regulatory roles of suppressor of cytokine signaling 5 (SOCS5) in T-ALL cellular signaling networks and leukemia progression. We found that SOCS5 was differentially expressed in primary T-ALL and its expression levels were lowered in HOXA-deregulated leukemia harboring KMT2A gene rearrangements. Here, we report that SOCS5 expression is epigenetically regulated by DNA methyltransferase-3A-mediated DNA methylation and methyl CpG binding protein-2-mediated histone deacetylation. We show that SOCS5 negatively regulates T-ALL cell growth and cell cycle progression but has no effect on apoptotic cell death. Mechanistically, SOCS5 silencing induces activation of JAK-STAT signaling, and negatively regulates interleukin-7 and interleukin-4 receptors. Using a human T-ALL murine xenograft model, we show that genetic inactivation of SOCS5 accelerates leukemia engraftment and progression, and leukemia burden. We postulate that SOCS5 is epigenetically deregulated in T-ALL and serves as an important regulator of T-ALL cell proliferation and leukemic progression. Our results link aberrant downregulation of SOCS5 expression to the enhanced activation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL.

Transcriptomes define distinct subgroups of salivary gland adenoid cystic carcinoma with different driver mutations and outcomes.

The relative rarity of salivary gland adenoid cystic carcinoma (ACC) and its slow growing yet aggressive nature has complicated the development of molecular markers for patient stratification. To analyze molecular differences linked to the protracted disease course of ACC and metastases that form 5 or more years after diagnosis, detailed RNA-sequencing (RNA-seq) analysis was performed on 68 ACC tumor samples, starting with archived, formalin-fixed paraffin-embedded (FFPE) samples up to 25 years old, so that clinical outcomes were available. A statistical peak-finding approach was used to classify the tumors that expressed MYB or MYBL1, which had overlapping gene expression signatures, from a group that expressed neither oncogene and displayed a unique phenotype. Expression of MYB or MYBL1 was closely correlated to the expression of the SOX4 and EN1 genes, suggesting that they are direct targets of Myb proteins in ACC tumors. Unsupervised hierarchical clustering identified a subgroup of approximately 20% of patients with exceptionally poor overall survival (median less than 30 months) and a unique gene expression signature resembling embryonic stem cells. The results provide a strategy for stratifying ACC patients and identifying the high-risk, poor-outcome group that are candidates for personalized therapies.

Optimized approach for Ion Proton RNA sequencing reveals details of RNA splicing and editing features of the transcriptome.

RNA-sequencing (RNA-seq) has become the standard method for unbiased analysis of gene expression but also provides access to more complex transcriptome features, including alternative RNA splicing, RNA editing, and even detection of fusion transcripts formed through chromosomal translocations. However, differences in library methods can adversely affect the ability to recover these different types of transcriptome data. For example, some methods have bias for one end of transcripts or rely on low-efficiency steps that limit the complexity of the resulting library, making detection of rare transcripts less likely. We tested several commonly used methods of RNA-seq library preparation and found vast differences in the detection of advanced transcriptome features, such as alternatively spliced isoforms and RNA editing sites. By comparing several different protocols available for the Ion Proton sequencer and by utilizing detailed bioinformatics analysis tools, we were able to develop an optimized random primer based RNA-seq technique that is reliable at uncovering rare transcript isoforms and RNA editing features, as well as fusion reads from oncogenic chromosome rearrangements. The combination of optimized libraries and rapid Ion Proton sequencing provides a powerful platform for the transcriptome analysis of research and clinical samples.

Distinct histone methylation and transcription profiles are established during the development of cellular quiescence in yeast.

BACKGROUND: Quiescent cells have a low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we defined the genome-wide profiles of three species of histone methylation associated with active transcription between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and transcripts. RESULTS: Quiescent cells retained histone methylations normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained significant levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The RNA polymerase II associated with genes in quiescent cells displayed a distinct occupancy profile compared to its pattern of occupancy across genes in actively growing cells. Although transcription is generally repressed in quiescent cells, analysis of individual genes identified a period of active transcription during the development of quiescence. CONCLUSIONS: The data suggest that the transcript profile and histone methylation marks in quiescent cells were established both in growing cells and during the development of quiescence and then retained in these cells. Together, this might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth.

Mutant-Allele Tumor Heterogeneity Scores Correlate With Risk of Metastases in Colon Cancer.

BACKGROUND: Colorectal cancer is a leading cause of cancer-related mortality, has a very broad mutational spectrum, and there is no clinically available biomarker that can predict which patients with stage II or stage III colorectal cancer will develop metastatic disease. PATIENTS AND METHODS: We used a targeted next-generation sequencing approach to analyze the mutational spectra in stage II and III colon cancer patient samples. RESULTS: Amidst a broad range of acquired mutations and variants, we found evidence of tumor heterogeneity that distinguished the tumors in different groups. When heterogeneity was quantified using the Mutant-Allele Tumor Heterogeneity (MATH) score, there was a strong correlation between higher MATH score and risk of metastases. CONCLUSIONS: Measures of tumor heterogeneity might be useful biomarkers for identifying patients with colon cancer who are at risk of developing metastases. This might allow for more specific, tailored follow-up and adjuvant therapies after standard surgery.

Integration of ruxolitinib into dose-intensified therapy targeted against a novel JAK2 F694L mutation in B-precursor acute lymphoblastic leukemia.

A 17-year-old girl with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with persistent minimal residual disease (MRD) who underwent standard chemotherapy was found to have a BCR-ABL1-like gene expression pattern. Genome sequencing revealed a JAK2 mutation not previously described in BCP-ALL and a potential therapeutic target. Due to concern for an on-therapy relapse, the JAK2 inhibitor ruxolitinib was incorporated into a modified chemotherapy backbone to achieve complete remission prior to stem cell transplant. Treatment was well tolerated and she had undetectable MRD prior to a matched allogeneic stem cell transplant and remained in remission at day +100.

Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma.

UNLABELLED: Adenoid cystic carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with a bleak prognosis for which therapeutic targets are unavailable. We used RNA sequencing (RNA-seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples. In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. RNA-seq provided information about gene fusions, alternative RNA splicing, and gene expression signatures. Interestingly, tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related MYB proteins are interchangeable oncogenic drivers in ACC. Our results provide important details about the biology of ACC and illustrate how archival tissue samples can be used for detailed molecular analyses of rare tumors. SIGNIFICANCE: Using RNA-seq to perform whole-transcriptome analysis of archival ACC tumor samples, we identified novel, recurrent gene fusions, detected alternative RNA splicing, and established gene expression signatures that provide detailed information about the biology of ACC tumors.

Situational awareness: regulation of the myb transcription factor in differentiation, the cell cycle and oncogenesis.

This review summarizes the mechanisms that control the activity of the c-Myb transcription factor in normal cells and tumors, and discusses how c-Myb plays a role in the regulation of the cell cycle. Oncogenic versions of c-Myb contribute to the development of leukemias and solid tumors such as adenoid cystic carcinoma, breast cancer and colon cancer. The activity and specificity of the c-Myb protein seems to be controlled through changes in protein-protein interactions, so understanding how it is regulated could lead to the development of novel therapeutic strategies.

RNA editing events in mitochondrial genes by ultra-deep sequencing methods: a comparison of cytoplasmic male sterile, fertile and restored genotypes in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in failure to produce functional pollen and is widely used in the production of hybrid seed. Improper RNA editing is implicated as the molecular basis for some CMS systems. However, the mechanism of CMS in cotton is unknown. This study compared RNA editing events in eight mitochondrial genes (atp1, 4, 6, 8, 9, and cox1, 2, 3) among three lines (maintainer B, CMS A, and restorer R). These events were quantified by ultra-deep sequencing of mitochondrial transcripts and sequencing of cloned versions of these genes as cDNAs. A comparison of genomic PCR and RT-PCR products detected 72 editing sites in coding sequences in the eight genes and four partial editing sites in the 3'-untranslated region of atp6. The most frequent alteration (61.4 %) resulted in changes of hydrophilic amino acids to hydrophobic amino acids and the most common alteration was proline (P) to leucine (L) (26.7 %). In atp6, RNA editing created a stop codon from a glutamine in the genomic sequence. Statistical analysis of the frequencies of RNA editing events detected differences between mtDNA genes, but no differences between cotton cytoplasms that could account for the CMS phenotype or restoration. This study represents the first work to use next-generation sequencing to identify RNA editing positions and efficiency, and possible association with CMS and restoration in plants.

Single molecule analysis of c-myb alternative splicing reveals novel classifiers for precursor B-ALL.

The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB) has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.