Clonal hematopoiesis in patients with HIV and cancer.

BACKGROUND: Cancer-related deaths for people living with HIV (PWH) are increasing due to longer life expectancies and disparately poor cancer-related outcomes. We hypothesize that advanced biological aging contributes to cancer-related morbidity and mortality for PWH and cancer. We sought to determine the impact of clonal hematopoiesis (CH) on cancer disparities in PWH. METHODS: We conducted a retrospective study to compare the prevalence and clinical outcomes of CH in PWH and people without HIV (PWoH) and cancer. Included in the study were PWH and similar PWoH based on tumor site, age, tumor sequence, and cancer treatment status. Biological aging was also measured using epigenetic methylation clocks. RESULTS: In 136 patients with cancer, PWH had twice the prevalence of CH compared to similar PWoH (23% vs 11%, p=0.07). After adjusting for patient characteristics, PWH were four-times more likely to have CH than PWoH (OR 4.1, 95% CI 1.3-13.9, p=0.02). The effect of CH on survival was most pronounced in PWH, who had a 5-year survival rate of 38% if they had CH (vs 59% if no CH), compared to PWoH who had a 5-year survival rate of 75% if they had CH (vs 83% if no CH). CONCLUSION: This study provides the first evidence that PWH may have a higher prevalence of CH than PWoH with the same cancers. CH may be an independent biological aging risk factor contributing to inferior survival for PWH and cancer.

The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss.

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare and aggressive malignancy. Though the molecular underpinnings of this cancer have been largely unexplored, recurrent chromosomal breakpoints affecting a noncoding region on chr19q13, which includes the chromosome 19 microRNA cluster (C19MC), have been reported in several cases. We performed comprehensive molecular profiling on samples from 14 patients diagnosed with UESL. Congruent with prior reports, we identified structural variants in chr19q13 in 10 of 13 evaluable tumors. From whole transcriptome sequencing, we observed striking expressional activity of the entire C19MC region. Concordantly, in 7 of 7 samples undergoing miRNAseq, we observed hyperexpression of the miRNAs within this cluster to levels >100 fold compared to matched normal tissue or a non-C19MC amplified cancer cell line. Concurrent TP53 mutation or copy number loss was identified in all evaluable tumors with evidence of C19MC overexpression. We find that C19MC miRNAs exhibit significant negative correlation to TP53 regulatory miRNAs and K-Ras regulatory miRNAs. Using RNA-seq we identified that pathways relevant to cellular differentiation as well as mRNA translation machinery are transcriptionally enriched in UESL. In summary, utilizing a combination of next-generation sequencing and high-density arrays we identify the combination of C19MC hyperexpression via chromosomal structural event with TP53 mutation or loss as highly recurrent genomic features of UESL.

A microRNA Signature Identifies Patients at Risk of Barrett Esophagus Progression to Dysplasia and Cancer.

BACKGROUND: Progression of Barrett esophagus (BE) to esophageal adenocarcinoma occurs among a minority of BE patients. To date, BE behavior cannot be predicted on the basis of histologic features. AIMS: We compared BE samples that did not develop dysplasia or carcinoma upon follow-up of ≥ 7 years (BE nonprogressed [BEN]) with BE samples that developed carcinoma upon follow-up of 3 to 4 years (BE progressed [BEP]). METHODS: The NanoString nCounter miRNA assay was used to profile 24 biopsy samples of BE, including 13 BENs and 11 BEPs. Fifteen samples were randomly selected for miRNA prediction model training; nine were randomly selected for miRNA validation. RESULTS: Unpaired t tests with Welch's correction were performed on 800 measured miRNAs to identify the most differentially expressed miRNAs for cases of BEN and BEP. The top 12 miRNAs (P < .003) were selected for principal component analyses: miR-1278, miR-1301, miR-1304-5p, miR-517b-3p, miR-584-5p, miR-599, miR-103a-3p, miR-1197, miR-1256, miR-509-3-5p, miR-544b, miR-802. The 12-miRNA signature was first self-validated on the training dataset, resulting in 7 out of the 7 BEP samples being classified as BEP (100% sensitivity) and 7 out of the 8 BEN samples being classified as BEN (87.5% specificity). Upon validation, 4 out of the 4 BEP samples were classified as BEP (100% sensitivity) and 4 out of the 5 BEN samples were classified as BEN (80% specificity). Twenty-four samples were evaluated, and 22 cases were correctly classified. Overall accuracy was 91.67%. CONCLUSION: Using miRNA profiling, we have identified a 12-miRNA signature able to reliably differentiate cases of BEN from BEP.

Establishing a living biobank of patient-derived organoids of intraductal papillary mucinous neoplasms of the pancreas.

Pancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this preclinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention.

Development of Malignancy-Risk Gene Signature Assay for Predicting Breast Cancer Risk.

BACKGROUND: Breast cancer (BC) risk assessment models are statistical estimates based on patient characteristics. We developed a gene expression assay to assess BC risk using benign breast biopsy tissue. METHODS: A NanoString-based malignancy risk (MR) gene signature was validated for formalin-fixed paraffin-embedded (FFPE) tissue. It was applied to FFPE benign and BC specimens obtained from women who underwent breast biopsy, some of whom developed BC during follow-up to evaluate diagnostic capability of the MR signature. BC risk was calculated with MR score, Gail risk score, and both tests combined. Logistic regression and receiver operating characteristic curves were used to evaluate these 3 models. RESULTS: NanoString MR demonstrated concordance between fresh frozen and FFPE malignant samples (r = 0.99). Within the validation set, 563 women with benign breast biopsies from 2007 to 2011 were identified and followed for at least 5 y; 50 women developed BC (affected) within 5 y from biopsy. Three groups were compared: benign tissue from unaffected and affected patients and malignant tissue from affected patients. Kruskal-Wallis test suggested difference between the groups (P = 0.09) with trend in higher predicted MR score for benign tissue from affected patients before development of BC. Neither the MR signature nor Gail risk score were statistically different between affected and unaffected patients; combining both tests demonstrated best predictive value (AUC = 0.71). CONCLUSIONS: FFPE gene expression assays can be used to develop a predictive test for BC. Further investigation of the combined MR signature and Gail Model is required. Our assay was limited by scant cellularity of archived breast tissue.

Clonal haemopoiesis and therapy-related myeloid malignancies in elderly patients: a proof-of-concept, case-control study.

BACKGROUND: Clonal haemopoiesis of indeterminate potential (CHIP) is an age-associated genetic event linked to increased risk of primary haematological malignancies and increased all-cause mortality, but the prevalence of CHIP in patients who develop therapy-related myeloid neoplasms is unknown. We did this study to investigate whether chemotherapy-treated patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. METHODS: We did a nested, case-control, proof-of-concept study to compare the prevalence of CHIP between patients with cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). We identified cases from our internal biorepository of 123 357 patients who consented to participate in the Total Cancer Care biobanking protocol at Moffitt Cancer Center (Tampa, FL, USA) between Jan 1, 2006, and June 1, 2016. We included all individuals who were diagnosed with a primary malignancy, were treated with chemotherapy, subsequently developed a therapy-related myeloid neoplasm, and were 70 years or older at either diagnosis. For inclusion in this study, individuals must have had a peripheral blood or mononuclear cell sample collected before the diagnosis of therapy-related myeloid neoplasm. Controls were individuals who were diagnosed with a primary malignancy at age 70 years or older and were treated with chemotherapy but did not develop therapy-related myeloid neoplasms. Controls were matched to cases in at least a 4:1 ratio on the basis of sex, primary tumour type, age at diagnosis, smoking status, chemotherapy drug class, and duration of follow-up. We used sequential targeted and whole-exome sequencing and described clonal evolution in cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available. The primary endpoint of this study was the development of therapy-related myeloid neoplasm and the primary exposure was CHIP. FINDINGS: We identified 13 cases and 56 case-matched controls. The prevalence of CHIP in all patients (23 [33%] of 69 patients) was higher than has previously been reported in elderly individuals without cancer (about 10%). Cases had a significantly higher prevalence of CHIP than did matched controls (eight [62%] of 13 cases vs 15 [27%] of 56 controls, p=0·024; odds ratio 5·75, 95% CI 1·52-25·09, p=0·013). The most commonly mutated genes in cases with CHIP were TET2 (three [38%] of eight patients) and TP53(three [38%] of eight patients), whereas controls most often had TET2 mutations (six [40%] of 15 patients). In most (four [67%] of six patients) cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available, the mean allele frequency of CHIP mutations had expanded by the time of the therapy-related myeloid neoplasm diagnosis. However, a subset of paired samples (two [33%] of six patients) had CHIP mutations that decreased in allele frequency, giving way to expansion of a distinct mutant clone. INTERPRETATION: Patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. The distribution of CHIP-related gene mutations differs between individuals with therapy-related myeloid neoplasm and those without, suggesting that mutation-specific differences might exist in therapy-related myeloid neoplasm risk. FUNDING: Moffitt Cancer Center.

Increased incidence of FBXW7 and POLE proofreading domain mutations in young adult colorectal cancers.

BACKGROUND: The incidence and outcomes of patients with colorectal cancer (CRC) varies by age. Younger patients tend to have sporadic cancers that are not detected by screening and worse survival. To understand whether genetic differences exist between age cohorts, the authors sought to characterize unique genetic alterations in patients with CRC. METHODS: In total, 283 patients who were diagnosed with sporadic CRC between 1998 and 2010 were identified and divided by age into 2 cohorts-ages ≤45 years (the younger cohort) and ≥65 years (the older cohort)-and targeted exome sequencing was performed. The Fisher exact test was used to detect differences in mutation frequencies between the 2 groups. Whole exome sequencing was performed on 21 additional younger patient samples for validation. Findings were confirmed in The Cancer Genome Atlas CRC data set. RESULTS: In total, 246 samples were included for final analysis (195 from the older cohort and 51 from the younger cohort). Mutations in the FBXW7 gene were more common in the younger cohort (27.5% vs 9.7%; P = .0022) as were mutations in the proofreading domain of polymerase ε catalytic subunit (POLE) (9.8% vs 1%; P = .0048). There were similar mutation rates between cohorts with regard to TP53 (64.7% vs 61.5%), KRAS (43.1% vs 46.2%), and APC (60.8% vs 73.8%). BRAF mutations were numerically more common in the older cohort, although the difference did not reach statistical significance (2% vs 9.7%; P = .082). CONCLUSIONS: In this retrospective study, a unique genetic profile was identified for younger patients who have CRC compared with patients who are diagnosed at an older age. These findings should be validated in a larger study and could have an impact on future screening and treatment modalities for younger patients with CRC. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2828-2835. © 2016 American Cancer Society.

Fibrosis and subsequent cytopenias are associated with basic fibroblast growth factor-deficient pluripotent mesenchymal stromal cells in large granular lymphocyte leukemia.

Cytopenias occur frequently in systemic lupus erythematosus, rheumatoid arthritis, Felty's syndrome, and large granular lymphocyte (LGL) leukemia, but the bone marrow microenvironment has not been systematically studied. In LGL leukemia (n = 24), retrospective analysis of bone marrow (BM) histopathology revealed severe fibrosis in 15 of 24 patients (63%) in association with the presence of cytopenias, occurrence of autoimmune diseases, and splenomegaly, but was undetectable in control cases with B cell malignancies (n = 11). Fibrosis severity correlated with T cell LGL cell numbers in the BM, but not in the periphery, suggesting deregulation is limited to the BM microenvironment. To identify fibrosis-initiating populations, primary mesenchymal stromal cultures (MSCs) from patients were characterized and found to display proliferation kinetics and overabundant collagen deposition, but displayed normal telomere lengths and osteoblastogenic, chondrogenic, and adipogenic differentiation potentials. To determine the effect of fibrosis on healthy hematopoietic progenitor cells (HPCs), bioartificial matrixes from rat tail or purified human collagen were found to suppress HPC differentiation and proliferation. The ability of patient MSCs to support healthy HSC proliferation was significantly impaired, but could be rescued with collagenase pretreatment. Clustering analysis confirmed the undifferentiated state of patient MSCs, and pathway analysis revealed an inverse relationship between cell division and profibrotic ontologies associated with reduced basic fibroblast growth factor production, which was confirmed by ELISA. Reconstitution with exogenous basic fibroblast growth factor normalized patient MSC proliferation, collagen deposition, and HPC supportive function, suggesting LGL BM infiltration and secondary accumulation of MSC-derived collagen is responsible for hematopoietic failure in autoimmune-associated cytopenias in LGL leukemia.

The functional role of L-fucose on dendritic cell function and polarization.

Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from melanoma- and breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

Implementation of a High-Accuracy Targeted Gene Expression Panel for Clinical Care.

This study describes the validation of a clinical RNA expression panel with evaluation of concordance between gene copy gain by a next-generation sequencing (NGS) assay and high gene expression by an RNA expression panel. The RNA Salah Targeted Expression Panel (RNA STEP) was designed with input from oncologists to include 204 genes with utility for clinical trial prescreening and therapy selection. RNA STEP was validated with the nanoString platform using remnant formalin-fixed, paraffin-embedded-derived RNA from 102 patients previously tested with a validated clinical NGS panel. The repeatability, reproducibility, and concordance of RNA STEP results with NGS results were evaluated. RNA STEP demonstrated high repeatability and reproducibility, with excellent correlation (r > 0.97, P < 0.0001) for all comparisons. Comparison of RNA STEP high gene expression (log2 ratio ≥ 2) versus NGS DNA-based gene copy number gain (copies ≥ 5) for 38 mutually covered genes revealed an accuracy of 93.0% with a positive percentage agreement of 69.4% and negative percentage agreement of 93.8%. Moderate correlation was observed between platforms (r = 0.53, P < 0.0001). Concordance between high gene expression and gene copy number gain varied by specific gene, and some genes had higher accuracy between assays. Clinical implementation of RNA STEP provides gene expression data complementary to NGS and offers a tool for prescreening patients for clinical trials.

Association between CYP3A4, CYP3A5 and ABCB1 genotype and tacrolimus treatment outcomes among allogeneic HSCT patients.

Aim: Successful treatment with tacrolimus to prevent graft versus host disease (GVHD) and minimize tacrolimus-related toxicities among allogeneic hematopoietic cell transplantation (alloHCT) recipients is contingent upon quickly achieving and maintaining concentrations within a narrow therapeutic range. The primary objective was to investigate associations between CYP3A4, CYP3A5 or ABCB1 genotype and the proportion of patients that attained an initial tacrolimus goal concentration following initiation of intravenous (iv.) and conversion to oral administration. Materials & methods: We retrospectively evaluated 86 patients who underwent HLA-matched (8/8) related donor alloHCT and were prescribed a tacrolimus-based regimen for GVHD prophylaxis. Results & conclusion: The findings of the present study suggests that CYP3A5 genotype may impact attainment of initial therapeutic tacrolimus concentrations with oral administration in alloHCT recipients.

Elevated Methionine Flux Drives Pyroptosis Evasion in Persister Cancer Cells.

Induction of cell death represents a primary goal of most anticancer treatments. Despite the efficacy of such approaches, a small population of "persisters" develop evasion strategies to therapy-induced cell death. While previous studies have identified mechanisms of resistance to apoptosis, the mechanisms by which persisters dampen other forms of cell death, such as pyroptosis, remain to be elucidated. Pyroptosis is a form of inflammatory cell death that involves formation of membrane pores, ion gradient imbalance, water inflow, and membrane rupture. Herein, we investigate mechanisms by which cancer persisters resist pyroptosis, survive, then proliferate in the presence of tyrosine kinase inhibitors (TKI). Lung, prostate, and esophageal cancer persister cells remaining after treatments exhibited several hallmarks indicative of pyroptosis resistance. The inflammatory attributes of persisters included chronic activation of inflammasome, STING, and type I interferons. Comprehensive metabolomic characterization uncovered that TKI-induced pyroptotic persisters display high methionine consumption and excessive taurine production. Elevated methionine flux or exogenous taurine preserved plasma membrane integrity via osmolyte-mediated effects. Increased dependency on methionine flux decreased the level of one carbon metabolism intermediate S-(5'-adenosyl)-L-homocysteine, a determinant of cell methylation capacity. The consequent increase in methylation potential induced DNA hypermethylation of genes regulating metal ion balance and intrinsic immune response. This enabled thwarting TKI resistance by using the hypomethylating agent decitabine. In summary, the evolution of resistance to pyroptosis can occur via a stepwise process of physical acclimation and epigenetic changes without existing or recurrent mutations. SIGNIFICANCE: Methionine enables cancer cells to persist by evading pyroptotic osmotic lysis, which leads to genome-wide hypermethylation that allows persisters to gain proliferative advantages.

Associations between prediagnostic aspirin use and ovarian tumor gene expression.

BACKGROUND: Aspirin use has been associated with reduced ovarian cancer risk, yet the underlying biological mechanisms are not fully understood. To gain mechanistic insights, we assessed the association between prediagnosis low and regular-dose aspirin use and gene expression profiles in ovarian tumors. METHODS: RNA sequencing was performed on high-grade serous, poorly differentiated, and high-grade endometrioid ovarian cancer tumors from the Nurses' Health Study (NHS), NHSII, and New England Case-Control Study (n = 92 cases for low, 153 cases for regular-dose aspirin). Linear regression identified differentially expressed genes associated with aspirin use, adjusted for birth decade and cohort. False discovery rates (FDR) were used to account for multiple testing and gene set enrichment analysis was used to identify biological pathways. RESULTS: No individual genes were significantly differentially expressed in ovarian tumors in low or regular-dose aspirin users accounting for multiple comparisons. However, current versus never use of low-dose aspirin was associated with upregulation of immune pathways (e.g., allograft rejection, FDR = 5.8 × 10-10 ; interferon-gamma response, FDR = 2.0 × 10-4 ) and downregulation of estrogen response pathways (e.g., estrogen response late, FDR = 4.9 × 10-8 ). Ovarian tumors from current regular aspirin users versus never users were also associated with upregulation in interferon pathways (FDR <1.5 × 10-4 ) and downregulation of multiple extracellular matrix (ECM) architecture pathways (e.g., ECM organization, 4.7 × 10-8 ). CONCLUSION: Our results suggest low and regular-dose aspirin may impair ovarian tumorigenesis in part via enhancing adaptive immune response and decreasing metastatic potential supporting the likely differential effects on ovarian carcinogenesis and progression by dose of aspirin.

Modeling phenotypic heterogeneity towards evolutionarily inspired osteosarcoma therapy.

Osteosarcoma is the most common bone sarcoma in children and young adults. While universally delivered, chemotherapy only benefits roughly half of patients with localized disease. Increasingly, intratumoral heterogeneity is recognized as a source of therapeutic resistance. In this study, we develop and evaluate an in vitro model of osteosarcoma heterogeneity based on phenotype and genotype. Cancer cell populations vary in their environment-specific growth rates and in their sensitivity to chemotherapy. We present the genotypic and phenotypic characterization of an osteosarcoma cell line panel with a focus on co-cultures of the most phenotypically divergent cell lines, 143B and SAOS2. Modest environmental (pH, glutamine) or chemical perturbations dramatically shift the success and composition of cell lines. We demonstrate that in nutrient rich culture conditions 143B outcompetes SAOS2. But, under nutrient deprivation or conventional chemotherapy, SAOS2 growth can be favored in spheroids. Importantly, when the simplest heterogeneity state is evaluated, a two-cell line coculture, perturbations that affect the faster growing cell line have only a modest effect on final spheroid size. Thus the only evaluated therapies to eliminate the spheroids were by switching therapies from a first strike to a second strike. This extensively characterized, widely available system, can be modeled and scaled to allow for improved strategies to anticipate resistance in osteosarcoma due to heterogeneity.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. METHODS: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT. RESULTS: We discovered that neoantigen-specific TIL clones were predominantly CD4+ T cells and were present in both therapeutic responders and non-responders. CD4+ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4+ TIL. CONCLUSIONS: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4+ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4+ TIL in future ACT protocols as a strategy to improve antitumor immunity.

Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM is regulated at the level of transcription, we resuspended cells that were cultivated under high-DIC conditions in chemostats in growth medium with low concentrations of DIC and tracked CCM development in the presence and absence of the RNA polymerase inhibitor rifampin. Induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by quantitative reverse transcription-PCR (qRT-PCR) and transmission electron microscopy, respectively. Genome-wide transcription patterns for cells grown under DIC limitation and those grown under ammonia limitation were assayed via microarrays and compared. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315) present in other organisms, including chemolithoautotrophs, but whose function(s) has not been elucidated in any organism were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and P(II) gene transcription, the transcription of two novel genes (Tcr_0466 and Tcr_2018) was measurably enhanced. Upregulation of all four genes (Tcr_1019, 4-fold; Tcr_131, ∼7-fold; Tcr_0466, >200-fold; Tcr_2018, 7-fold), which suggests that novel components are part of the response to nutrient limitation by this organism, was verified via qRT-PCR.

Lifetime ovulatory years and ovarian cancer gene expression profiles.

BACKGROUND: Greater ovulatory years is associated with increased ovarian cancer risk. Although ovulation leads to an acute pro-inflammatory local environment, how long-term exposure to ovulation impacts ovarian carcinogenesis is not fully understood. Thus, we examined the association between gene expression profiles of ovarian tumors and lifetime ovulatory years to enhance understanding of associated biological pathways. METHODS: RNA sequencing data was generated on 234 invasive ovarian cancer tumors that were high-grade serous, poorly differentiated, or high-grade endometrioid from the Nurses' Health Study (NHS), NHSII, and the New England Case Control Study. We used linear regression to identify differentially expressed genes by estimated ovulatory years, adjusted for birth decade and cohort, overall and stratified by menopausal status at diagnosis. We used false discovery rates (FDR) to account for multiple testing. Gene set enrichment analysis (GSEA) with Cancer Hallmarks, KEGG, and Reactome databases was used to identify biological pathways associated with ovulatory years. RESULTS: No individual genes were significantly differentially expressed by ovulatory years (FDR > 0.19). However, GSEA identified several pathways that were significantly associated with ovulatory years, including downregulation of pathways related to inflammation and proliferation (FDR < 1.0 × 10-5). Greater ovulatory years were more strongly associated with downregulation of genes related to proliferation (e.g., E2F targets, FDR = 1.53 × 10-24; G2M checkpoints, FDR = 3.50 × 10-22) among premenopausal versus postmenopausal women at diagnosis. The association of greater ovulatory years with downregulation of genes involved in inflammatory response such as interferon gamma response pathways (FDR = 7.81 × 10-17) was stronger in postmenopausal women. CONCLUSIONS: Our results provide novel insight into the biological pathways that link ovulatory years to ovarian carcinogenesis, which may lead to development of targeted prevention strategies for ovarian cancer.

T cell repertoire in peripheral blood as a potential biomarker for predicting response to concurrent cetuximab and nivolumab in head and neck squamous cell carcinoma.

BACKGROUND: T cell receptor (TCR) signaling profile is a fundamental property that underpins both adaptive and innate immunity in the host. Despite its potential clinical relevance, the TCR repertoire in peripheral blood has not been thoroughly explored for its value as an immunotherapy efficacy biomarker in head and neck squamous cell carcinoma (HNSCC). The purpose of the present study is to characterize and compare the TCR repertoire in peripheral blood mononuclear cells (PBMC) from patients with HNSCC treated with the combination of cetuximab and nivolumab. METHODS: We used the immunoSEQ assay to sequence the TCR beta (TCR-B) chain repertoire from serially obtained PBMC at baseline and during the treatments from a total of 41 patients who received the combination (NCT03370276). Key TCR repertoire metrics, including diversity and clonality, were calculated and compared between patients with different therapy responses and clinical characteristics (eg, human papillomavirus (HPV) status and smoking history). Patient survival outcomes were compared according to patient groups stratified by the TCR-B clonotyping. To confirm the observed patterns in TCR spectrum, samples from patients who achieved complete response (CR) and partial response (PR) were further profiled with the immunoSEQ deep resolution assay. RESULTS: Our data indicated that the patients who achieved CR and PR had an increased TCR sequence diversity in their baseline samples, this tendency being more pronounced in HPV-negative patients or those with a smoking history. Notably, the CR/PR group had the lowest proportion of patients with oligoclonal TCR clones (2 out of 8 patients), followed by the stable disease group (9 out of 20 patients) and lastly the progressive disease group (7 out of 10 patients). An overall trend toward favorable patient survival was also observed in the polyclonal group. Finally, we reported the shared TCR clones across patients within the same response group, as well as the shared clones by aligning immunoSEQ reads with TCR data retrieved from The Cancer Genome Atlas- head and neck squamous cell carcinoma (TCGA-HNSC) cohort. CONCLUSIONS: Our data suggest that, despite the great clinical heterogeneity of HNSCC and the limited responders in the present cohort, the peripheral TCR repertoires from pretreatment PBMC may be developed as biomarkers for the benefit of immunotherapy in HNSCC.