High Level Immunoglobulin Gene Expression and Reduced Intra-tumoral Bacteria Associated With the Transition from Initial to Progressive Diffuse Intrinsic Pontine Glioma.

BACKGROUND/AIM: In the past decade, diffuse intrinsic pontine glioma (DIPG), the most common childhood brainstem glioma, has benefitted from an increase in tissue-based research because of improved biopsy collection techniques. However, the adaptive immune receptor (IR) features represented by tumor material and tumor infiltrating lymphocytes have remained poorly understood. MATERIALS AND METHODS: Herein, we characterized the adaptive immune parameters of DIPG through the recovery of IR recombination reads from RNAseq files representing initial and progressive DIPG samples. RESULTS: An elevated level of immunoglobulin gene expression in the progressive DIPG sample files and a reduced number of bacterial sequencing read recoveries in comparison to RNAseq files representing the initial form of DIPG, was found. Furthermore, the RNAseq files representing both initial and progressive DIPG samples had significant numbers of reads representing Cutibacterium acnes, a bacterium previously linked to prostate cancer development. Results also indicated an opportunity to distinguish overall survival probabilities based on IGL complementarity determining region-3 amino acid sequence physicochemical parameters. CONCLUSION: Genomics analyses allow for a better understanding of adaptive IR features and bacterial infections in the DIPG setting.

TCR CDR3-CMV Antigen Chemical Complementaries Are Associated With a Worse Outcome for Renal Cell Carcinoma.

BACKGROUND/AIM: Due to still unresolved questions regarding viruses as either a primary cause or a comorbidity in cancer, we examined a potential immune response to cytomegalovirus (CMV) in the renal cell carcinoma (RCC) setting using genomics and bioinformatics approaches. MATERIALS AND METHODS: Specifically, we assessed chemical complementarity scores (CSs) for solid tissue normal resident, T-cell receptor (TCR) complementarity determining region 3 (CDR3s) and CMV antigens and determined whether higher or lower CS groups were associated with a higher or lower survival probability. RESULTS: This was indeed the case, with all such analyses consistently indicating a lower overall and progression-free survival for the cases representing the higher TCR CDR3-CMV antigen chemical CSs. This basic result was obtained for two separate RCC datasets and multiple CMV antigens. CONCLUSION: The results raise the question, to what extent a systemic CMV infection may represent an important co-morbidity for RCC.

IGL CDR3 Hydropathy and Antigen Chemical Complementarity Associated with Greater Disease-Free Survival in Lung Adenocarcinoma: Implications for Gender Disparities.

With lung cancer remaining a challenging disease, new approaches to biomarker discovery and therapy development are needed. Recent immunogenomics, adaptive immune receptor approaches have indicated that it is very likely that B cells play an important role in mediating better overall outcomes. As such, we assessed physicochemical features of lung adenocarcinoma resident IGL complementarity determining region-3 (CDR3) amino acid (AA) sequences and determined that hydrophobic CDR3 AA sequences were associated with a better disease-free survival (DFS) probability. Further, using a recently developed chemical complementarity scoring algorithm particularly suitable for the evaluation of large patient datasets, we determined that IGL CDR3 chemical complementarity with certain cancer testis antigens was associated with better DFS. Chemical complementarity scores for IGL CDR3-MAGEC1 represented a gender bias, with an overrepresentation of males among the higher IGL-CDR3-CTA complementarity scores that were in turn associated with better DFS (logrank p < 0.065). Overall, this study pointed towards potential biomarkers for prognoses that, in some cases are likely gender-specific; and towards biomarkers for guiding therapy, e.g., IGL-based opportunities for antigen targeting in the lung cancer setting.

Chemical Complementarity of Tumor Resident, Adaptive Immune Receptor CDR3s and Previously Defined Hepatitis C Virus Epitopes Correlates with Improved Outcomes in Hepatocellular Carcinoma.

To better understand how adaptive immune receptors (IRs) in hepatocellular carcinoma (HCC) microenvironments are related to disease outcomes, we employed a chemical complementarity scoring algorithm to quantify electrostatic complementarity between HCC tumor TRB or IGH complementarity-determining region 3 (CDR3) amino acid (AA) sequences and previously characterized hepatitis C virus (HCV) epitopes. High electrostatic complementarity between HCC-resident CDR3s and 12 HCV epitopes was associated with greater survival probabilities, as indicated by two distinct HCC IR CDR3 datasets. Two of the HCV epitopes, HCV*71871 (TRB) and HCV*13458 (IGH), were also determined to represent significantly larger electrostatic CDR3-HCV epitope complementarity in HCV-positive HCC cases, compared with HCV-negative HCC cases, with the CDR3s representing yet a third, independent HCC dataset. Overall, the results indicated the utility of CDR3 AA sequences as biomarkers for HCC patient stratification and as potential guides for the development of therapeutic reagents.

TRB CDR3 chemical complementarity with HBV epitopes correlates with increased hepatocellular carcinoma, disease-free survival.

The liver is a site of immune privilege, compared with the bladder and skin, for example. To study this attenuation of the immune response in the cancer setting, we compared quantities and features of adaptive immune receptor (IR) recombination reads obtained from hepatocellular carcinoma (HCC) and six other cancers. Of these cancers, HCC had the lowest numbers of IR recombination reads and was the only cancer with a greater number immunoglobulin rather than T-cell receptor recombination reads. To better understand the role of adaptive IRs obtained from the tumor microenvironment in shaping the outcome of HCC cases, we quantified the chemical complementarity between HCC tumor TRB and IGH complementarity determining region-3 (CDR3) amino acid (AA) sequences, and known hepatitis B virus (HBV) epitopes. High chemical complementarity between HCC-resident CDR3s and three HBV epitopes correlated with increased survival probabilities, for two sources of CDR3s representing different CDR3 recovery algorithms. These results suggest the potential of CDR3 AA sequences as biomarkers for HCC patient stratification and as guides for future development of therapeutics.

Exploiting big data survival information to unify risk-stratification related, adaptive immune receptor parameters for multiple myeloma.

With the improvement of treatment options, multiple myeloma related life expectancy has been prolonged, but the disease remains largely incurable. Immunotherapy is a growing field that shows promise in advancements for treatment, and recent work has demonstrated an opportunity to use immune receptor, complementarity determining region-3 (CDR3)-candidate antigen chemical complementarity scores to identify survival distinctions among subgroups of patients. Here, we have applied the complementarity scoring algorithm to identify multiple myeloma related, CDR3-cancer testis antigen (CTA) relationships associated with survival distinctions. Furthermore, we have overlapped these immune receptor features with a previous study that showed a dramatic survival distinction based on T-cell receptor, V- and J-gene segment usage, HLA allele combinations, whereby 100% of the patients in certain combination groups had no mortality related to multiple myeloma, during the study period. This overlap evaluation was consistent with the idea that there are likely considerable constraints on productive TRB-antigen-HLA combinations but more flexibility, and unpredictability, for the TRA-antigen-HLA combinations. Also, the approaches in this reported indicated the potential importance of the CTA, IGSF11, as a multiple myeloma antigen, an antigen previously, independently considered as a vaccine candidate in other settings.

Chemical features of melanoma tumor resident TRG CDR3s associated with better survival probabilities.

We assessed the T-cell receptor gamma (TRG) recombination reads from the cancer genome atlas melanoma tumor exome files and the TRG recombination reads from an independent, melanoma exome file dataset, from the Moffitt Cancer Center. TRG complementarity determining region-3 (CDR3) amino acid (AA) sequences were assessed for chemical complementarity to cancer testis antigens, with such complementarity for FAM133A and CRISP2 associated with better survival probabilities for both datasets. These results, along with related TRG CDR3 AA chemical feature assessments provided in this report, have indicated opportunities for melanoma patient stratifications based on the recovery of TRG recombination reads from both tumor and blood samples, and the results may point towards novel, effective melanoma antigens.

The presence of intratumoral Porphyromonas gingivalis correlates with a previously defined pancreatic adenocarcinoma, immune cell expression phenotype and with tumor resident, adaptive immune receptor features.

The association between pancreatic adenocarcinoma (PAAD) and the pancreatic microbiome is not fully understood, although bacteria may decrease the effectiveness of chemotherapy and lead to anti-apoptotic, pro-inflammatory microenvironments. To better understand the relationship between the PAAD microbiome and the microenvironment, we identified Porphyromonas gingivalis-positive PAAD samples and found a strong association between intratumoral P. gingivalis and: (i) an immune cell gene expression phenotype previously defined by others as gene program 7; and (ii) recovery of immunoglobulin recombination, sequencing reads. We applied a novel chemical complementarity scoring algorithm, suitable for a big data setting, and determined that the previously established P. gingivalis antigen, rpgB had a reduced chemical complementarity with T-cell receptor (TCR) complementarity-determining region-3 amino acid sequences recovered from PAAD samples with P. gingivalis in comparison to TCR-rpgB chemical complementarity represented by the PAAD samples that lacked P. gingivalis. This finding strengthens the existing body of evidence correlating P. gingivalis with PAAD, which may have implications for the treatment and prognosis of patients. Furthermore, demonstrating the correlation of P. gingivalis and gene program 7 raises the question of whether P. gingivalis infection is responsible for the gene program 7 subdivision of PAAD?

Bacterial Sequencing Reads in Blood Exome Files from Melanoma and Cervical Cancer Patients are Associated with Cancer Recurrence.

Bacteremia poses great risk for morbidity and mortality for immunocompromised cancer patients. Although the presence of bacteria within solid tumors is gaining greater attention, few studies have analyzed species of bacteria in the blood and their effect on cancer clinical outcomes. Using the Kraken 2 taxonomic profiling tool, we classified bacteria present in blood and primary tumors of cervical cancer and melanoma cases. The Cancer Genome Atlas (TCGA) melanoma blood exome files with Pseudomonas species were found to represent a worse disease-free survival (DFS) probability, while a worse overall survival (OS) result was evidenced for both the TCGA and Moffitt Cancer Center melanoma datasets. Cervical cancer cases with reads representing the Bradyrhizobium genus and Bradyrhizobium sp. BTAi1 found in blood and tumor exome files were found to have lower DFS. Additionally, reduced DFS and OS were observed for cervical cancer cases positive for Bacteroides species including Bacteroides fragilis. This study provides novel evidence and a novel approach for indicating that bacteria in blood is associated with cancer recurrence. These findings may guide the development of more efficient prognostic and screening tools related to bacterial blood infections of melanoma and cervical cancer patients.

TCR gene segment usage and HLA alleles that are associated with cancer survival rates also represent racial disparities.

Understanding racial disparities in cancer outcomes continues to be a challenge, with likely many factors at play, including socioeconomic factors and genetic polymorphisms impacting basic cellular and molecular functions. Additionally, it is possible that specific combinations of environment and genetics have specific impacts. T-cell receptor (TCR) gene segment usage, HLA allele combinations have been associated with autoimmune and infectious disease courses, and more recently, TCR gene segment usage, HLA allele combinations have been associated with distinct survival outcomes in cancer as well. We examined several such, previously reported cancer-related TCR gene segment usage, HLA allele combinations for evidence of racial disparities, with regard to the prevalence of the combination in different racial groups. Results indicated that TCR gene segment usage, potentially reflecting environmental factors related to previous pathogen exposure, in combination with certain HLA alleles or independently, may represent a novel explanation for racial disparities in cancer outcomes. Overall, at this point, a genetic connection to racial disparities in cancer outcomes is detectable but remains modest, suggesting that other factors, such as socioeconomic factors, remain as important considerations.

Chemical complementarity between tumor resident, T-cell receptor CDR3s and MAGEA3/6 correlates with increased melanoma survival: Potential relevance to MAGE vaccine auto-reactivity.

Cancer testis antigens have been of interest as possible targets for cancer immunotherapies. To better understand the opportunities for the use of such immunotherapy targets, we used a chemical complementarity scoring algorithm and an original web tool to establish aspects of electrostatic complementarity of the CTAs, MAGEA3 and MAGEA6, with melanoma specimen resident, T-cell receptor (TCR) complementarity determining region 3 (CDR3) amino acid sequences. Greater electrostatic complementarity between T-cell receptor CDR3 and tumor CTAs MAGEA3/6 was associated with a greater probability of overall survival, for both the cancer genome atlas and Moffitt Cancer Center samples; and was associated with high levels of T-cell cytotoxicity-related gene expression. Most importantly, this approach allowed for the highly efficient screening of specific segments of the MAGEA3/6 antigens which indicated that certain MAGE segments would have either more or less risk of auto-reactivity. In sum, the chemical complementarity algorithm, and its efficient application via the web tool, adaptivematch.com, offers a convenient opportunity to identify likely parameters important for immunotherapy considerations and melanoma patient risk stratifications.

Immunogenomics Parameters for Patient Stratification in Alzheimer's Disease.

BACKGROUND: Despite the fact that only modest adaptive immune system related approaches to treating Alzheimer's disease (AD) are available, an immunogenomics approach to the study of AD has not yet substantially advanced. OBJECTIVE: Thus, we sought to better understand adaptive immune receptor chemical features in the AD setting. METHODS: We characterized T-cell receptor alpha (TRA) complementarity determining region-3 (CDR3) physicochemical features and identified TRA CDR3 homology groups, represented by TRA recombination reads extracted from 2,665 AD-related, blood- and brain-derived exome files. RESULTS: We found that a higher isoelectric value for the brain TRA CDR3s was associated with a higher (clinically worse) Braak stage and that a number of TRA CDR3 chemical homology groups, in particular representing bloodborne TRA CDR3s, were associated with higher or lower Braak stages. Lastly, greater chemical complementarity of both blood- and brain-derived TRA CDR3s and tau, based on a recently described CDR3-candidate antigen chemical complementarity scoring process (https://adaptivematch.com), was associated with higher Braak stages. CONCLUSION: Overall, the data reported here raise the questions of (a) whether progression of AD is facilitated by the adaptive immune response to tau; and (b) whether assessment of such an anti-tau immune response could potentially serve as a basis for adaptive immune receptor related, AD risk stratification?

Specific TCR V-J gene segment recombinations leading to the identification pan-V-J CDR3s associated with survival distinctions: diffuse large B-cell lymphoma.

In the diffuse large B-cell lymphoma (DLBCL) setting, we examined lymph node biopsy, T-cell receptor features, and the DLBLC patient human leukocyte antigen (HLA) alleles, to provide a basis for assessing survival distinctions represented by the National Cancer Institute Center for Cancer Research (NCICCR) dataset. While previous analyses of other cancer datasets have indicated that specific T-cell receptor (TCR) V or J gene segments, independently, can be associated with a survival distinction, we have here identified V-J recombinations, representing specific V and J gene segments associated with survival distinctions. As specific V-J recombinations represent relatively conserved complementarity determining region-3 (CDR3) amino acid sequences, we assessed the entire DLBCL NCICCR dataset for such conserved CDR3 features. Overall, this approach indicated the opportunity of identifying DLBCL patient subpopulations with TCR CDR3 features, and HLA alleles, with significant survival distinctions, possibly identifying cohorts more likely to benefit from a given immunotherapy.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

High-throughput, sliding-window algorithm for assessing chemical complementarity between immune receptor CDR3 domains and cancer mutant peptides: TRG-PIK3CA interactions and breast cancer.

Physicochemical assessments of a vast accumulation of adaptive immune receptor (IR) recombinations have led to correlations of those properties with sub-divisions of various diseases. In the cancer setting, such assessments, particularly for the complementarity determining region-3 (CDR3) immune receptor domain, have been used to establish chemical complementarity matches to mutant amino acids (AA). These matches, in some cases, over very large numbers of tumor samples, have correlated with survival and gene expression distinctions. For example, in melanoma, electrostatic charge based, T-cell receptor CDR3-DNAH9 mutant AA complementarity represents better survival over multiple datasets that represent tumor tissue, T-cell receptor CDR3s. In this report, the complementarity approach has been expanded to include a more comprehensive representation of the interaction of T-cell receptor CDR3s and mutant AAs by incorporating the impact of the wild-type AAs surrounding the mutant AA. This "sliding window" approach was benchmarked against two large datasets of empirically determined CDR3-epitope pairs; showed more significant patient subdivisions; revealed a novel, TRG CDR3-mutant PIK3CA linkage in breast cancer; and was particularly suited to use with big data collections using only modest and widely-available processors. Thus, the algorithm should support more rapid and convenient indications (or prescreens) of CDR3-mutant peptide interactions for more focused studies and more efficient development of patient immunology-related prognostic tools and therapies.

Chemical complementarity between immune receptors and cancer mutants, independent of antigen presentation protein binding, is associated with increased survival rates.

Uterine cancer has been associated with a T-cell immune response that leads to increased survival. Therefore, we used several bioinformatics approaches to explore specific interactions between T-cell receptor (TCR) and tumor mutant peptide sequences. Using endometrioid uterine cancer exome files from the The Cancer Genome Atlas database, we obtained tumor resident V-J recombinations for the T-Cell Receptor alpha gene (TRA). The charged-based, chemical complementarity for each patient's LRP2 or TTN mutant amino acids (AAs) and the recovered, TRA complementarity determining region-3 (CDR3) sequences was calculated, allowing a division of patients into complementary and noncomplementary groups. Complementary groups with TTN mutants had increased disease-free survival and increased expression of complement genes. Furthermore, the survival distinction based on CDR3-mutant peptide complementarity was independent of programmatically assessed HLA class II binding and was not observable based on the CDR3 AA chemical features alone. The above approach provides a potential, highly efficient method for identifying TCR targets in uterine cancer and may aid in the development of novel prognostic tools.

Specific HLA alleles, paired with TCR V- and J-gene segment usage, link to distinct multiple myeloma survival rates.

Multiple myeloma (MM) immunogenomics studies related to T-cell characterizations and involving large patient sets have been lacking, particularly in comparison to solid tumor types. Thus, we evaluated (i) HLA alleles, and (ii) T-Cell Receptor (TCR) V- and J-gene segment, HLA allele combinations, based on TCR recombinations in blood samples, for their potential associations with overall survival distinctions among an MM cohort. Two HLA alleles, and seven TCR V- or J-gene segment, HLA allele combinations were found to be associated with distinct overall survival rates. For examples, HLA-C*08:02, and the TRAV19, HLA-C*07:01 combination, were found to be associated with negative outcomes. In addition, anti-cytomegalovirus immune receptor sequences, from blood samples, were found to be associated with a positive outcome (p = 0.012, n = 278). These data, and other related immunogenomics data, indicate a potential opportunity to use personal immunogenetics parameters as guides to prognosis and therapies.

Electrostatic Complementarity of T-Cell Receptor-Alpha CDR3 Domains and Mutant Amino Acids Is Associated with Better Survival Rates for Sarcomas.

While sarcoma immunology has advanced with regard to basic, and even some applied topics, this disease has not been subject to more recent immunogenomics approaches. Thus, we assessed the immune receptor recombinations available from the cancer genome atlas (TCGA) sarcoma database via tumor sample exome and RNASeq files. Results indicated that recovery of T-cell receptor-alpha recombination reads (TRA) correlated with a better survival rate, with the expression of T-cell biomarkers, and with tumor sample apoptosis signatures consistent with the longer patient survival times. Furthermore, samples representing TRA complementarity determining region-3 (CDR3) net charge per residue (NCPR) based complementarity with the corresponding sarcoma mutanome had a better survival rate, and more granzyme expression, than samples lacking such complementarity. By specifically using RNASeq-recovered TRA CDR3s and related NCPR assessments, three genes, TP53, ATRX, and RB1, were identified as being key components of the mutanome-based complementarity. Thus, these genes may represent key immune system targets for soft tissue sarcomas. Also, several key results from above were reproduced with a pediatric osteosarcoma dataset, work that led to identification of MUC6 mutations as potentially linked to a strong immune response. In sum, TRA CDR3s are likely to be important prognostic indicators, and possibly a beginning tool for immunotherapy development strategies, for adult and pediatric sarcomas.