Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma.

Radiation therapy is frequently used to treat cancers including soft tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in transplanted tumors, but the mechanism(s) remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, we performed bulk RNA-seq, single-cell RNA-seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and interferon-γ. CpG + RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG + RT, TCR clonality analysis suggests an increase in clonal T-cell dominance. Collectively, these findings demonstrate that RT + CpG significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft tissue sarcoma.

Temporary Knockdown of p53 During Focal Limb Irradiation Increases the Development of Sarcomas.

Approximately half of patients with cancer receive radiotherapy and, as cancer survivorship increases, the low rate of radiation-associated sarcomas is rising. Pharmacologic inhibition of p53 has been proposed as an approach to ameliorate acute injury of normal tissues from genotoxic therapies, but how this might impact the risk of therapy-induced cancer and normal tissue injuries remains unclear. We utilized mice that express a doxycycline (dox)-inducible p53 short hairpin RNA to reduce Trp53 expression temporarily during irradiation. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were subjected to RNA sequencing. Following single high-dose irradiation, 21% of animals with temporary p53 knockdown during irradiation developed a sarcoma in the radiation field compared with 2% of control animals. Following high-dose irradiation, p53 knockdown preserves muscle stem cells, and increases sarcoma development. Mice with severe acute radiation-induced injuries exhibit an increased risk of developing late persistent wounds, which were associated with sarcomagenesis. RNA sequencing revealed radiation-induced sarcomas upregulate genes related to translation, epithelial-mesenchymal transition (EMT), inflammation, and the cell cycle. Comparison of the transcriptomes of human and mouse sarcomas that arose in irradiated tissues revealed regulation of common gene programs, including elevated EMT pathway gene expression. These results suggest that blocking p53 during radiotherapy could minimize acute toxicity while exacerbating late effects including second cancers. SIGNIFICANCE: Strategies to prevent or mitigate acute radiation toxicities include pharmacologic inhibition of p53 and other cell death pathways. Our data show that temporarily reducing p53 during irradiation increases late effects including sarcomagenesis.

Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include "shock and kill" strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

Shining a LIGHT on myeloid cell targeted immunotherapy.

Despite over a decade of clinical trials combining inhibition of emerging checkpoints with a PD-1/L1 inhibitor backbone, meaningful survival benefits have not been shown in PD-1/L1 inhibitor resistant or refractory solid tumours, particularly tumours dominated by a myelosuppressive microenvironment. Achieving durable anti-tumour immunity will therefore likely require combination of adaptive and innate immune stimulation, myeloid repolarisation, enhanced APC activation and antigen processing/presentation, lifting of the CD47/SIRPα (Cluster of Differentiation 47/signal regulatory protein alpha) 'do not eat me' signal, provision of an apoptotic 'pro-eat me' or 'find me' signal, and blockade of immune checkpoints. The importance of effectively targeting mLILRB2 and SIRPAyeloid cells to achieve improved response rates has recently been emphasised, given myeloid cells are abundant in the tumour microenvironment of most solid tumours. TNFSF14, or LIGHT, is a tumour necrosis superfamily ligand with a broad range of adaptive and innate immune activities, including (1) myeloid cell activation through Lymphotoxin Beta Receptor (LTßR), (2) T/NK (T cell and natural killer cell) induced anti-tumour immune activity through Herpes virus entry mediator (HVEM), (3) potentiation of proinflammatory cytokine/chemokine secretion through LTßR on tumour stromal cells, (4) direct induction of tumour cell apoptosis in vitro, and (5) the reorganisation of lymphatic tissue architecture, including within the tumour microenvironment (TME), by promoting high endothelial venule (HEV) formation and induction of tertiary lymphoid structures. LTBR (Lymphotoxin beta receptor) and HVEM rank highly amongst a range of costimulatory receptors in solid tumours, which raises interest in considering how LIGHT-mediated costimulation may be distinct from a growing list of immunotherapy targets which have failed to provide survival benefit as monotherapy or in combination with PD-1 inhibitors, particularly in the checkpoint acquired resistant setting.

CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.

Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include "shock and kill" strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

Pre-treatment differential correlation of gene expression and response to topical steroids in eosinophilic esophagitis.

Few predictors of response to topical corticosteroid (tCS) treatment have been identified in eosinophilic esophagitis (EoE). We aimed to determine whether baseline gene expression predicts histologic response to tCS treatment for EoE. We analyzed prospectively collected samples from incident EoE cases who were treated with tCS for 8 weeks in a development cohort (prospective study) or in an independent validation cohort (clinical trial). Whole transcriptome RNA expression was determined from a baseline (pre-treatment) RNA-later preserved esophageal biopsy. Baseline expression was compared between histologic responders (<15 eos/hpf) and non-responders (≥15 eos/hpf), and differential correlation was used to assess baseline gene expression by response status. In 87 EoE cases analyzed in the development set, there were no differentially expressed genes associated with treatment response (at false discovery rate = 0.1). However, differential correlation identified a module of 22 genes with statistically significantly high pairwise correlation in non-responders (mean correlation coefficient = 0.7) compared to low correlation in responders (coefficient = 0.3). When this 22-gene module was applied to the 89 EoE cases in the independent cohort, it was not validated to predict tCS response at the 15 eos/hpf threshold (mean correlation coefficient = 0.32 in responders and 0.25 in nonresponders). Exploration of other thresholds also did not validate any modules. Though we identified a 22 gene differential correlation module measured pre-treatment that was strongly associated with subsequent histologic response to tCS in EoE, this was not validated in an independent population. Alternative methods to predict steroid response should be explored.

Neoadjuvant Radiation Therapy and Surgery Improves Metastasis-Free Survival over Surgery Alone in a Primary Mouse Model of Soft Tissue Sarcoma.

This study aims to investigate whether adding neoadjuvant radiotherapy (RT), anti-programmed cell death protein-1 (PD-1) antibody (anti-PD-1), or RT + anti-PD-1 to surgical resection improves disease-free survival for mice with soft tissue sarcomas (STS). We generated a high mutational load primary mouse model of STS by intramuscular injection of adenovirus expressing Cas9 and guide RNA targeting Trp53 and intramuscular injection of 3-methylcholanthrene (MCA) into the gastrocnemius muscle of wild-type mice (p53/MCA model). We randomized tumor-bearing mice to receive isotype control or anti-PD-1 antibody with or without radiotherapy (20 Gy), followed by hind limb amputation. We used micro-CT to detect lung metastases with high spatial resolution, which was confirmed by histology. We investigated whether sarcoma metastasis was regulated by immunosurveillance by lymphocytes or tumor cell-intrinsic mechanisms. Compared with surgery with isotype control antibody, the combination of anti-PD-1, radiotherapy, and surgery improved local recurrence-free survival (P = 0.035) and disease-free survival (P = 0.005), but not metastasis-free survival. Mice treated with radiotherapy, but not anti-PD-1, showed significantly improved local recurrence-free survival and metastasis-free survival over surgery alone (P = 0.043 and P = 0.007, respectively). The overall metastasis rate was low (∼12%) in the p53/MCA sarcoma model, which limited the power to detect further improvement in metastasis-free survival with addition of anti-PD-1 therapy. Tail vein injections of sarcoma cells into immunocompetent mice suggested that impaired metastasis was due to inability of sarcoma cells to grow in the lungs rather than a consequence of immunosurveillance. In conclusion, neoadjuvant radiotherapy improves metastasis-free survival after surgery in a primary model of STS.

CALGB 40603 (Alliance): Long-Term Outcomes and Genomic Correlates of Response and Survival After Neoadjuvant Chemotherapy With or Without Carboplatin and Bevacizumab in Triple-Negative Breast Cancer.

PURPOSE: CALGB 40603 (NCT00861705), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS: The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS: Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION: Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

Genomic characterization of rare molecular subclasses of hepatocellular carcinoma.

Primary liver cancer, consisting of both cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC), is the second leading cause of cancer deaths worldwide. Our goal is to genomically characterize rare HCC subclasses to provide insight into disease biology. Leveraging The Cancer Genome Atlas (TCGA) to perform a combined analysis of CCA (n = 36) and HCC (n = 275), we integrated multiple genomic platforms, to assess transcriptional profiles, mutational signatures, and copy number patterns to uncover underlying etiology and linage specific patterns. We identified two molecular classes distinct from prototypical HCC tumors. The first, CCA-Like, although histologically indistinguishable from HCC, had enrichment of CCA mutations (IDH1, BAP1), mutational signatures, and transcriptional patterns (SOX9, KRT19). CCA-Like, however, retained a copy number landscape similar to HCC, suggesting a hepatocellular linage. The second, Blast-Like, is enriched in TP53 mutations, HBV infection, exposure related mutational signatures and transcriptionally similar to hepatoblasts. Although these subclasses are molecularly distinct, they both have a worse progression-free survival compared to classical HCC tumors, yet are clinically treated the same. The identification of and characterization of CCA-Like and Blast-Like subclasses advance our knowledge of HCC as well as represents an urgent need for the identification of class specific biomarkers and targeted therapy.

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors.

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.

Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague.

Following inhalation, Yersinia pestis rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in Yersinia spp., the factors mediating early Y. pestis adherence in the lung remain unknown. To identify genes important for Y. pestis adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δcaf1) Y. pestis transposon mutant libraries were serially passaged in vivo to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δcaf1Y. pestis backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for YPO3903 in Y. pestis adherence in the lung and showed that YPO3903 regulated transcript levels of psaA, which encodes a fimbria previously implicated in Y. pestis adherence in vitro Deletion of psaA had a minor effect on Y. pestis adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to psaA By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early Y. pestis adherence during primary pneumonic plague.IMPORTANCE Colonization of the lung by Yersinia pestis is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which Y. pestis adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify Y. pestis genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple Y. pestis adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing Y. pestis adherence and the subsequent development of pneumonic plague.

FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

Identifying transcriptional profiles and evaluating prognostic biomarkers of HIV-associated diffuse large B-cell lymphoma from Malawi.

Lymphoma incidence in sub-Saharan Africa (SSA) is increasing due to HIV and population aging. Diffuse Large B-cell lymphoma (DLBCL), the most common lymphoma in SSA and worldwide, is highly associated with HIV, but molecular studies of HIV-associated DLBCL are scarce globally. We describe profiling of DLBCL from Malawi, aiming to elucidate tumor biology and identify clinically meaningful biomarkers specifically for SSA. Between June 1, 2013 and June 1, 2016, 59 cases of DLBCL (32 HIV+/27 HIV-) enrolled in the Kamuzu Central Hospital Lymphoma Study were characterized, of which 54 (92%) were negative for Epstein-Barr virus. Gene expression profiling (GEP) by whole transcriptome sequencing was performed on the first 36 cases (22 HIV+/14 HIV-). Immunohistochemistry (IHC) and GEP results were compared with published data and correlated to clinical outcome and pathologic features. Unsupervised clustering strongly segregated DLBCL by HIV status (p = 0.0003, Chi-squared test), indicating a marked contribution of HIV to expression phenotype. Pathway analysis identified that HIV-associated tumors were enriched in hypoxia, oxidative stress, and metabolism related gene expression patterns. Cell-of-origin subtype, determined by sequencing and IHC, did not associate with differences in overall survival (OS), while Ki-67 proliferation index ≥80% was associated with inferior OS in HIV+ DLBCL only (p = 0.03) and cMYC/BCL2 co-expression by IHC was negatively prognostic across the entire cohort (p = 0.01). This study provides among the first molecular characterizations of DLBCL from SSA, demonstrates marked gene expression differences by HIV status, and identifies genomic and immunophenotypic characteristics that can inform future basic and clinical investigations.

Virus expression detection reveals RNA-sequencing contamination in TCGA.

BACKGROUND: Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequences. To detect viruses with high specificity, we developed the publicly available workflow, VirDetect, that detects virus and laboratory vector sequences in RNA-seq samples. We applied VirDetect to 9143 RNA-seq samples sequenced at one TCGA sequencing center (28/33 cancer types) over 5 years. RESULTS: We confirmed that H-HPV18 was present in many samples and determined that viral transcripts from H-HPV18 significantly co-occurred with those from xenotropic mouse leukemia virus-related virus (XMRV). Using laboratory metadata and viral transcription, we determined that the likely contaminant was a pool of cell lines known as the "common reference", which was sequenced alongside TCGA RNA-seq samples as a control to monitor quality across technology transitions (i.e. microarray to GAII to HiSeq), and to link RNA-seq to previous generation microarrays that standardly used the "common reference". One of the cell lines in the pool was a laboratory isolate of MCF-7, which we discovered was infected with XMRV; another constituent of the pool was likely HeLa cells. CONCLUSIONS: Altogether, this indicates a multi-step contamination process. First, MCF-7 was infected with an XMRV. Second, this infected cell line was added to a pool of cell lines, which contained HeLa. Finally, RNA from this pool of cell lines contaminated several TCGA tumor samples most-likely during library construction. Thus, these human tumors with H-HPV or XMRV reads were likely not infected with H-HPV 18 or XMRV.

RNA Expression Profiling of Lymphoepithelioma-Like Carcinoma of the Bladder Reveals a Basal-Like Molecular Subtype.

Lymphoepithelioma-like carcinoma of the bladder (LELC-B) is a rare subtype of urothelial carcinoma consisting of undifferentiated epithelial cells within a dense inflammatory cell infiltrate. We set out to molecularly characterize LELC-B through RNA expression profiling as well as immunohistochemistry (IHC) to understand its underlying biology. Sixteen cases of LELC-B were identified at Johns Hopkins University. RNA sequencing was performed on 14 cases. IHC staining for programmed cell death ligand 1 (PD-L1) and mismatch repair proteins MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, and PMS1 homolog, mismatch repair system component 2 (PMS2) was performed. Transcriptomic profiling of LELC-B showed that they are enriched in a basal-like phenotype, with 12 of 14 LELC-B cases correlating to the basal centroid of the bladder cancer analysis of subtypes by gene expression 47 (BASE47) predictive analysis of microarrays (PAM) classifier. Gene signature analysis confirmed the lymphocyte infiltration profile consistent with the histomorphology. LELC-B lacked features to explain the robust lymphocytic infiltrate, such as loss of mismatch repair protein expression or expression of Epstein-Barr virus transcripts. Nonetheless, PD-L1 IHC was positive in 93% of LELC cases. Our study demonstrates that LELC-B tumors are enriched in a basal-like molecular subtype and share a high level of immune infiltration and PD-L1 expression, similar to basal tumors. The basal-like phenotype is consistent with the known sensitivity of LELC-B to chemotherapy and suggests that immune checkpoint therapy should be explored in this rare disease.

An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma.

The incidence of human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is increasing and implicated in more than 60% of all oropharyngeal carcinomas (OPSCCs). Although whole-genome, transcriptome, and proteome analyses have identified altered signaling pathways in HPV-induced HNSCCs, additional tools are needed to investigate the unique pathobiology of OPSCC. Herein, bioinformatics analyses of human HPV(+) HNSCCs revealed that all tumors express full-length E6 and identified molecular subtypes based on relative E6 and E7 expression levels. To recapitulate the levels, stoichiometric ratios, and anatomic location of E6/E7 expression, we generated a genetically engineered mouse model whereby balanced expression of E6/E7 is directed to the oropharyngeal epithelium. The addition of a mutant PIK3CAE545K allele leads to the rapid development of pre-malignant lesions marked by immune cell accumulation, and a subset of these lesions progress to OPSCC. This mouse provides a faithful immunocompetent model for testing treatments and investigating mechanisms of immunosuppression.

Machine-Learning Prediction of Tumor Antigen Immunogenicity in the Selection of Therapeutic Epitopes.

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity.

Alternative tumour-specific antigens.

The study of tumour-specific antigens (TSAs) as targets for antitumour therapies has accelerated within the past decade. The most commonly studied class of TSAs are those derived from non-synonymous single-nucleotide variants (SNVs), or SNV neoantigens. However, to increase the repertoire of available therapeutic TSA targets, 'alternative TSAs', defined here as high-specificity tumour antigens arising from non-SNV genomic sources, have recently been evaluated. Among these alternative TSAs are antigens derived from mutational frameshifts, splice variants, gene fusions, endogenous retroelements and other processes. Unlike the patient-specific nature of SNV neoantigens, some alternative TSAs may have the advantage of being widely shared by multiple tumours, allowing for universal, off-the-shelf therapies. In this Opinion article, we will outline the biology, available computational tools, preclinical and/or clinical studies and relevant cancers for each alternative TSA class, as well as discuss both current challenges preventing the therapeutic application of alternative TSAs and potential solutions to aid in their clinical translation.

Prognostic value of B cells in cutaneous melanoma.

BACKGROUND: Measures of the adaptive immune response have prognostic and predictive associations in melanoma and other cancer types. Specifically, intratumoral T cell density and function have considerable prognostic and predictive value in skin cutaneous melanoma (SKCM). Less is known about the significance of tumor-infiltrating B cells in SKCM. Our goal was to understand the prognostic and predictive value of B cell phenotypic subsets in SKCM using RNA sequencing. METHODS: We used our previously published algorithm, V'DJer, to assemble B cell receptor (BCR) repertoires and estimate diversity from short-read RNA sequencing (RNA-seq). We applied machine learning-based cellular phenotype classifiers to measure relative similarity of bulk tumor sample gene expression profiles and different B cell phenotypes. We assessed these aspects of B cell biology in 473 SKCM from the Cancer Genome Atlas Project (TCGA) as well as in RNA-seq data corresponding to tumor samples procured from patients who received CTLA-4 and PD-1 inhibitors for metastatic SKCM. RESULTS: We found that the BCR repertoire was associated with different clinical factors, such as tumor tissue site and sex. However, increased clonality of the BCR repertoire was favorably prognostic in SKCM and was prognostic even after first conditioning on various clinical factors. Mutation burden was not correlated with any BCR measurement, and no specific mutation had an altered BCR repertoire. Lack of an assembled BCR in pre-treatment tumor tissues was associated with a lack of anti-tumor response to a CTLA-4 inhibitor in metastatic SKCM. CONCLUSIONS: These findings suggest an important prognostic and predictive role for B cell characteristics in SKCM. This has implications for melanoma immunobiology and potential development of immunogenomics features to predict survival and response to immunotherapy.