Pre-treatment peripheral blood immunophenotyping and response to neoadjuvant chemotherapy in operable breast cancer.

BACKGROUND: Tumor immune infiltration and peripheral blood immune signatures have prognostic and predictive value in breast cancer. Whether distinct peripheral blood immune phenotypes are associated with response to neoadjuvant chemotherapy (NAC) remains understudied. METHODS: Peripheral blood mononuclear cells from 126 breast cancer patients enrolled in a prospective clinical trial (NCT02022202) were analyzed using Cytometry by time-of-flight with a panel of 29 immune cell surface protein markers. Kruskal-Wallis tests or Wilcoxon rank-sum tests were used to evaluate differences in immune cell subpopulations according to breast cancer subtype and response to NAC. RESULTS: There were 122 evaluable samples: 47 (38.5%) from patients with hormone receptor-positive, 39 (32%) triple-negative (TNBC), and 36 (29.5%) HER2-positive breast cancer. The relative abundances of pre-treatment peripheral blood T, B, myeloid, NK, and unclassified cells did not differ according to breast cancer subtype. In TNBC, higher pre-treatment myeloid cells were associated with lower pathologic complete response (pCR) rates. In hormone receptor-positive breast cancer, lower pre-treatment CD8 + naïve and CD4 + effector memory cells re-expressing CD45RA (TEMRA) T cells were associated with more extensive residual disease after NAC. In HER2 + breast cancer, the peripheral blood immune phenotype did not differ according to NAC response. CONCLUSIONS: Pre-treatment peripheral blood immune cell populations (myeloid in TNBC; CD8 + naïve T cells and CD4 + TEMRA cells in luminal breast cancer) were associated with response to NAC in early-stage TNBC and hormone receptor-positive breast cancers, but not in HER2 + breast cancer. TRIAL REGISTRATION: NCT02022202 . Registered 20 December 2013.

T-cell phenotype including CD57+ T follicular helper cells in the tumor microenvironment correlate with a poor outcome in follicular lymphoma.

T-lymphocytes are prevalent in the tumor microenvironment of follicular lymphoma (FL). However, the phenotype of T-cells may vary, and the prevalence of certain T-cell subsets may influence tumor biology and patient survival. We therefore analyzed a cohort of 82 FL patients using CyTOF to determine whether specific T-cell phenotypes were associated with distinct tumor microenvironments and patient outcome. We identified four immune subgroups with differing T-cell phenotypes and the prevalence of certain T-cell subsets was associated with patient survival. Patients with increased T cells with early differentiation stage tended to have a significantly better survival than patients with increased T-cells of late differentiation stage. Specifically, CD57+ TFH cells, with a late-stage differentiation phenotype, were significantly more abundant in FL patients who had early disease progression and therefore correlated with an inferior survival. Single cell analysis (CITE-seq) revealed that CD57+ TFH cells exhibited a substantially different transcriptome from CD57- TFH cells with upregulation of inflammatory pathways, evidence of immune exhaustion and susceptibility to apoptosis. Taken together, our results show that different tumor microenvironments among FL patients are associated with variable T-cell phenotypes and an increased prevalence of CD57+ TFH cells is associated with poor patient survival.

Quantitative spatial evaluation of tumor-immune interactions in the immunotherapy setting of metastatic melanoma lymph nodes.

Introduction: Immune cell infiltration into the tumor microenvironment is generally associated with favorable clinical outcomes in solid tumors. However, the dynamic interplay among distinct immune cell subsets within the tumor-immune microenvironment as it relates to clinical responses to immunotherapy remains unresolved. In this study, we applied multiplex immunofluorescence (MxIF) to spatially characterize tumor-immune interactions within the metastatic melanoma lymph node. Methods: Pretreatment, whole lymph node biopsies were evaluated from 25 patients with regionally metastatic melanoma who underwent subsequent anti-PD1 therapy. Cyclic MxIF was applied to quantitatively and spatially assess expression of 45 pathologist-validated antibodies on a single tissue section. Pixel-based single cell segmentation and a supervised classifier approach resolved 10 distinct tumor, stromal and immune cell phenotypes and functional expression of PD1. Results: Single cell analysis across 416 pathologist-annotated tumor core regions of interest yielded 5.5 million cells for spatial evaluation. Cellular composition of tumor and immune cell subsets did not differ in the tumor core with regards to recurrence outcomes (p>0.05) however spatial patterns significantly differed in regional and paracrine neighborhood evaluations. Specifically, a regional community cluster comprised of primarily tumor and dendritic cells was enriched in patients that did not experience recurrence (p=0.009). By an independent spatial approach, cell-centric neighborhood analyses identified an enrichment for dendritic cells in cytotoxic T cell (CTL) and tumor cell-centric neighborhoods in the no recurrence patient response group (p<0.0001). Further evaluation of these neighborhoods identified an enrichment for CTL-dendritic cell interactions in patients that did not experience recurrence (p<0.0001) whereas CTL-macrophage interactions were more prevalent in CTL-centric neighborhoods of patients who experienced recurrence (p<0.0001). Discussion: Overall, this study offers a more comprehensive evaluation of immune infiltrates and spatial-immune signatures in the metastatic tumor-immune microenvironment as it informs recurrence risk following immunotherapy.

Resolving the heterogeneous tumor-centric cellular neighborhood through multiplexed, spatial paracrine interactions in the setting of immune checkpoint blockade.

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion including the downregulation of HLA-I potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially-preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence (MxIF) to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune and vasculature components across 323 fields of view. Single cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core FOVs of responders. Integrating single cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the TCCN based on anatomical subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytical framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.

Towards defining morphologic parameters of normal parous and nulliparous breast tissues by artificial intelligence.

BACKGROUND: Breast terminal duct lobular units (TDLUs), the source of most breast cancer (BC) precursors, are shaped by age-related involution, a gradual process, and postpartum involution (PPI), a dramatic inflammatory process that restores baseline microanatomy after weaning. Dysregulated PPI is implicated in the pathogenesis of postpartum BCs. We propose that assessment of TDLUs in the postpartum period may have value in risk estimation, but characteristics of these tissues in relation to epidemiological factors are incompletely described. METHODS: Using validated Artificial Intelligence and morphometric methods, we analyzed digitized images of tissue sections of normal breast tissues stained with hematoxylin and eosin from donors ≤ 45 years from the Komen Tissue Bank (180 parous and 545 nulliparous). Metrics assessed by AI, included: TDLU count; adipose tissue fraction; mean acini count/TDLU; mean dilated acini; mean average acini area; mean "capillary" area; mean epithelial area; mean ratio of epithelial area versus intralobular stroma; mean mononuclear cell count (surrogate of immune cells); mean fat area proximate to TDLUs and TDLU area. We compared epidemiologic characteristics collected via questionnaire by parity status and race, using a Wilcoxon rank sum test or Fisher's exact test. Histologic features were compared between nulliparous and parous women (overall and by time between last birth and donation [recent birth: ≤ 5 years versus remote birth: > 5 years]) using multivariable regression models. RESULTS: Normal breast tissues of parous women contained significantly higher TDLU counts and acini counts, more frequent dilated acini, higher mononuclear cell counts in TDLUs and smaller acini area per TDLU than nulliparas (all multivariable analyses p < 0.001). Differences in TDLU counts and average acini size persisted for > 5 years postpartum, whereas increases in immune cells were most marked ≤ 5 years of a birth. Relationships were suggestively modified by several other factors, including demographic and reproductive characteristics, ethanol consumption and breastfeeding duration. CONCLUSIONS: Our study identified sustained expansion of TDLU numbers and reduced average acini area among parous versus nulliparous women and notable increases in immune responses within five years following childbirth. Further, we show that quantitative characteristics of normal breast samples vary with demographic features and BC risk factors.

The proteome of distal nerves: implication in delayed repair and poor functional recovery.

Chronic denervation is one of the key factors that affect nerve regeneration. Chronic axotomy deteriorates the distal nerve stump, causes protein changes, and renders the microenvironment less permissive for regeneration. Some of these factors/proteins have been individually studied. To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect, we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model. Four rats that received immediate repair after sciatic nerve transection served as control, whereas four rats in the experimental group (chronic denervation) had their sciatic nerve repaired after a 12-week delay. All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins. Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels. NanoLC-MS/MS analysis was applied to the gels. Protein expression levels of nerves on the surgery side were compared to those on the contralateral side. Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed. All the differentially expressed proteins in both groups were further stratified according to the biological processes. A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration. Ingenuity Pathway Analysis (IPA) software was used for pathway analysis. The results showed that 709 differentially expressed proteins were identified in the delayed repair group, with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified. The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated. IPA revealed that protective pathways such as LXR/RXR, acute phase response, RAC, ERK/MAPK, CNTF, IL-6, and FGF signaling were inhibited in the delayed repair group, whereas three detrimental pathways, including the complement system, PTEN, and apoptosis signaling, were activated. An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types. The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

Evaluation of circulating cell-free DNA in cholestatic liver disease using liver-specific methylation markers.

BACKGROUND: Quantification of circulating organ-specific cell-free DNA (cfDNA) provides a sensitive measure of ongoing cell death that could benefit evaluation of the cholestatic liver diseases primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), which lack reliable non-invasive biomarkers. Our goal in this pilot study was to determine whether liver-specific cfDNA levels are increased in PBC and PSC patients relative to controls and in advanced versus early disease, to evaluate their potential as novel disease biomarkers. METHODS: Peripheral blood derived bisulfite-treated DNA was PCR amplified from patients with PBC (n = 48), PSC (n = 48) and controls (n = 96) to evaluate methylation status at 16 CpG sites reported to be specifically unmethylated in liver tissue near the genes IGF2R, ITIH4 and VTN. Amplicons were used to prepare paired end libraries which were sequenced on a MiSeq sequencer. Trimmed reads were aligned and used to determine unmethylation ratios and to calculate concentration of liver-specific cfDNA. Comparisons between groups were performed using the two-tailed Mann-Whitney Test and relationships between variables were evaluated using Pearson's Correlation. RESULTS: Levels of liver-specific cfDNA, as measured at the 3 genetic loci, were increased in PBC and PSC patients relative to controls and in late-stage relative to early-stage patients. As well, cfDNA levels were correlated with levels of alkaline phosphatase, a commonly used biochemical test to evaluate disease severity in liver disease, in patients, but not in controls. CONCLUSIONS: cfDNA offers promise as a non-invasive liquid-biopsy to evaluate liver-specific cell-death in patients with cholestatic liver diseases.

Genome-wide resolution peripheral blood methylome profiling reveals signatures for cholestatic liver disease.

Aim: To profile DNA methylation changes of primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Materials & methods: Patients with: PBC, PSC with inflammatory bowel disease (IBD), PSC without IBD, and age-, sex-matched controls were profiled for methylomes of peripheral blood by reduced representation bisulfite sequencing. Differentially methylated CpG (DMC) and differentially methylated region (DMR) were detected and compared. Results: We identified consistently altered DMCs and DMRs across diseases with involvement in key pathways. Many similarities noted between two subtypes of PSC, interestingly few existed between PBC and PSC. DMRs were highly enriched with transcription factor binding. Top DMC changes were validated in liver tissue of an independent cohort. Conclusion: Methylome profiling provides insights to PBC and PSC.

Comprehensive annotation of BRCA1 and BRCA2 missense variants by functionally validated sequence-based computational prediction models.

PURPOSE: To improve methods for predicting the impact of missense variants of uncertain significance (VUS) in BRCA1 and BRCA2 on protein function. METHODS: Functional data for 248 BRCA1 and 207 BRCA2 variants from assays with established high sensitivity and specificity for damaging variants were used to recalibrate 40 in silico algorithms predicting the impact of variants on protein activity. Additional random forest (RF) and naïve voting method (NVM) metapredictors for both BRCA1 and BRCA2 were developed to increase predictive accuracy. RESULTS: Optimized thresholds for in silico prediction models significantly improved the accuracy of predicted functional effects for BRCA1 and BRCA2 variants. In addition, new BRCA1-RF and BRCA2-RF metapredictors showed area under the curve (AUC) values of 0.92 (95% confidence interval [CI]: 0.88-0.96) and 0.90 (95% CI: 0.84-0.95), respectively. Similarly, the BRCA1-NVM and BRCA2-NVM models had AUCs of 0.93 and 0.90. The RF and NVM models were used to predict the pathogenicity of all possible missense variants in BRCA1 and BRCA2. CONCLUSION: The recalibrated algorithms and new metapredictors significantly improved upon current models for predicting the impact of variants in cancer risk-associated domains of BRCA1 and BRCA2. Prediction of the functional impact of all possible variants in BRCA1 and BRCA2 provides important information about the clinical relevance of variants in these genes.

Triple-Negative Breast Cancer Risk Genes Identified by Multigene Hereditary Cancer Panel Testing.

BACKGROUND: Germline genetic testing with hereditary cancer gene panels can identify women at increased risk of breast cancer. However, those at increased risk of triple-negative (estrogen receptor-negative, progesterone receptor-negative, human epidermal growth factor receptor-negative) breast cancer (TNBC) cannot be identified because predisposition genes for TNBC, other than BRCA1, have not been established. The aim of this study was to define the cancer panel genes associated with increased risk of TNBC. METHODS: Multigene panel testing for 21 genes in 8753 TNBC patients was performed by a clinical testing laboratory, and testing for 17 genes in 2148 patients was conducted by a Triple Negative Breast Cancer Consortium (TNBCC) of research studies. Associations between deleterious mutations in cancer predisposition genes and TNBC were evaluated using results from TNBC patients and reference controls. RESULTS: Germline pathogenic variants in BARD1, BRCA1, BRCA2, PALB2, and RAD51D were associated with high risk (odds ratio > 5.0) of TNBC and greater than 20% lifetime risk for overall breast cancer among Caucasians. Pathogenic variants in BRIP1, RAD51C, and TP53 were associated with moderate risk (odds ratio > 2) of TNBC. Similar trends were observed for the African American population. Pathogenic variants in these TNBC genes were detected in 12.0% (3.7% non-BRCA1/2) of all participants. CONCLUSIONS: Multigene hereditary cancer panel testing can identify women with elevated risk of TNBC due to mutations in BARD1, BRCA1, BRCA2, PALB2, and RAD51D. These women can potentially benefit from improved screening, risk management, and cancer prevention strategies. Patients with mutations may also benefit from specific targeted therapeutic strategies.

Evaluation of ACMG-Guideline-Based Variant Classification of Cancer Susceptibility and Non-Cancer-Associated Genes in Families Affected by Breast Cancer.

Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of a variant-classification methodology based on American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG-designated reportable cancer and non-cancer-associated genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole-exome sequencing in 404 individuals in 253 families and classified 1,640 variants. Potentially clinically actionable (likely pathogenic [LP] or pathogenic [P]) versus nonactionable (VUS, likely benign, or benign) calls were 95% concordant with locus-specific databases and Clinvar. LP or P mutations were identified in 12 of 25 breast cancer susceptibility genes in 26 families without identified BRCA1/2 mutations (11%). Evaluation of 84 additional genes associated with autosomal-dominant cancer susceptibility identified LP or P mutations in only two additional families (0.8%). However, individuals from 10 of 253 families (3.9%) had incidental LP or P mutations in 32 non-cancer-associated genes, and 9% of individuals were monoallelic carriers of a rare LP or P mutation in 39 genes associated with autosomal-recessive cancer susceptibility. Furthermore, 95% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant-classification guidelines. Additionally, evaluation of extended panels of cancer-associated genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results.

Label-Free Proteomic Analysis of Protein Changes in the Striatum during Chronic Ethanol Use and Early Withdrawal.

The molecular mechanisms underlying the neuronal signaling changes in alcohol addiction and withdrawal are complex and multifaceted. The cortico-striatal circuit is highly implicated in these processes, and the striatum plays a significant role not only in the early stages of addiction, but in the developed-addictive state as well, including withdrawal symptoms. Transcriptional analysis is a useful method for determining changes in gene expression, however, the results do not always accurately correlate with protein levels. In this study, we employ label-free proteomic analysis to determine changes in protein expression within the striatum during chronic ethanol use and early withdrawal. The striatum, composed primarily of medium spiny GABAergic neurons, glutamatergic and dopaminergic nerve terminals and astrocytes, is relatively homogeneous for proteomic analysis. We were able to analyze more than 5000 proteins from both the dorsal (caudate and putamen) and ventral (nucleus accumbens) striatum and identified significant changes following chronic intermittent ethanol exposure and acute (8 h) withdrawal compared to ethanol naïve and ethanol exposure groups respectively. Our results showed significant changes in proteins involved in glutamate and opioid peptide signaling, and also uncovered novel pathways including mitochondrial function and lipid/cholesterol metabolism, as revealed by changes in electron transport chain proteins and RXR activation pathways. These results will be useful in the development of novel treatments for alcohol withdrawal and thereby aid in recovery from alcohol use disorder.

Prevalence of Pathogenic Mutations in Cancer Predisposition Genes among Pancreatic Cancer Patients.

The prevalence of germline pathogenic mutations in a comprehensive panel of cancer predisposition genes is not well-defined for patients with pancreatic ductal adenocarcinoma (PDAC). To estimate the frequency of mutations in a panel of 22 cancer predisposition genes, 96 patients unselected for a family history of cancer who were recruited to the Mayo Clinic Pancreatic Cancer patient registry over a 12-month period were screened by next-generation sequencing. Fourteen pathogenic mutations in 13 patients (13.5%) were identified in eight genes: four in ATM, two in BRCA2, CHEK2, and MSH6, and one in BARD1, BRCA1, FANCM, and NBN. These included nine mutations (9.4%) in established pancreatic cancer genes. Three mutations were found in patients with a first-degree relative with PDAC, and 10 mutations were found in patients with first- or second-degree relatives with breast, pancreas, colorectal, ovarian, or endometrial cancers. These results suggest that a substantial proportion of patients with PDAC carry germline mutations in predisposition genes associated with other cancers and that a better understanding of pancreatic cancer risk will depend on evaluation of families with broad constellations of tumors. These findings highlight the need for recommendations governing germline gene-panel testing of patients with pancreatic cancer.

PANDA: pathway and annotation explorer for visualizing and interpreting gene-centric data.

Objective. Bringing together genomics, transcriptomics, proteomics, and other -omics technologies is an important step towards developing highly personalized medicine. However, instrumentation has advances far beyond expectations and now we are able to generate data faster than it can be interpreted. Materials and Methods. We have developed PANDA (Pathway AND Annotation) Explorer, a visualization tool that integrates gene-level annotation in the context of biological pathways to help interpret complex data from disparate sources. PANDA is a web-based application that displays data in the context of well-studied pathways like KEGG, BioCarta, and PharmGKB. PANDA represents data/annotations as icons in the graph while maintaining the other data elements (i.e., other columns for the table of annotations). Custom pathways from underrepresented diseases can be imported when existing data sources are inadequate. PANDA also allows sharing annotations among collaborators. Results. In our first use case, we show how easy it is to view supplemental data from a manuscript in the context of a user's own data. Another use-case is provided describing how PANDA was leveraged to design a treatment strategy from the somatic variants found in the tumor of a patient with metastatic sarcomatoid renal cell carcinoma. Conclusion. PANDA facilitates the interpretation of gene-centric annotations by visually integrating this information with context of biological pathways. The application can be downloaded or used directly from our website: http://bioinformaticstools.mayo.edu/research/panda-viewer/.

The Biological Reference Repository (BioR): a rapid and flexible system for genomics annotation.

MOTIVATION: The Biological Reference Repository (BioR) is a toolkit for annotating variants. BioR stores public and user-specific annotation sources in indexed JSON-encoded flat files (catalogs). The BioR toolkit provides the functionality to combine and retrieve annotation from these catalogs via the command-line interface. Several catalogs from commonly used annotation sources and instructions for creating user-specific catalogs are provided. Commands from the toolkit can be combined with other UNIX commands for advanced annotation processing. We also provide instructions for the development of custom annotation pipelines. AVAILABILITY AND IMPLEMENTATION: The package is implemented in Java and makes use of external tools written in Java and Perl. The toolkit can be executed on Mac OS X 10.5 and above or any Linux distribution. The BioR application, quickstart, and user guide documents and many biological examples are available at http://bioinformaticstools.mayo.edu.