Novel autoantibodies help diagnose anti-SSA antibody negative Sjögren disease and predict abnormal labial salivary gland pathology.

OBJECTIVES: Sjögren disease (SjD) diagnosis often requires either positive anti-SSA antibodies or a labial salivary gland biopsy with a positive focus score (FS). One-third of patients with SjD lack anti-SSA antibodies (SSA-), requiring a positive FS for diagnosis. Our objective was to identify novel autoantibodies to diagnose 'seronegative' SjD. METHODS: IgG binding to a high-density whole human peptidome array was quantified using sera from SSA- SjD cases and matched non-autoimmune controls. We identified the highest bound peptides using empirical Bayesian statistical filters, which we confirmed in an independent cohort comprising SSA- SjD (n=76), sicca-controls without autoimmunity (n=75) and autoimmune-feature controls (SjD features but not meeting SjD criteria; n=41). In this external validation, we used non-parametric methods for binding abundance and controlled false discovery rate in group comparisons. For predictive modelling, we used logistic regression, model selection methods and cross-validation to identify clinical and peptide variables that predict SSA- SjD and FS positivity. RESULTS: IgG against a peptide from D-aminoacyl-tRNA deacylase (DTD2) bound more in SSA- SjD than sicca-controls (p=0.004) and combined controls (sicca-controls and autoimmune-feature controls combined; p=0.003). IgG against peptides from retroelement silencing factor-1 and DTD2 were bound more in FS-positive than FS-negative participants (p=0.010; p=0.012). A predictive model incorporating clinical variables showed good discrimination between SjD versus control (area under the curve (AUC) 74%) and between FS-positive versus FS-negative (AUC 72%). CONCLUSION: We present novel autoantibodies in SSA- SjD that have good predictive value for SSA- SjD and FS positivity.

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Natural History of Colorectal Polyps Undergoing Longitudinal in Vivo CT Colonography Surveillance.

Background The natural history of colorectal polyps is not well characterized due to clinical standards of care and other practical constraints limiting in vivo longitudinal surveillance. Established CT colonography (CTC) clinical screening protocols allow surveillance of small (6-9 mm) polyps. Purpose To assess the natural history of colorectal polyps followed with CTC in a clinical screening program, with histopathologic correlation for resected polyps. Materials and Methods In this retrospective study, CTC was used to longitudinally monitor small colorectal polyps in asymptomatic adult patients from April 1, 2004, to August 31, 2020. All patients underwent at least two CTC examinations. Polyp growth patterns across multiple time points were analyzed, with histopathologic context for resected polyps. Regression analysis was performed to evaluate predictors of advanced histopathology. Results In this study of 475 asymptomatic adult patients (mean age, 56.9 years ± 6.7 [SD]; 263 men), 639 unique polyps (mean initial diameter, 6.3 mm; volume, 50.2 mm3) were followed for a mean of 5.1 years ± 2.9. Of these 639 polyps, 398 (62.3%) underwent resection and histopathologic evaluation, and 41 (6.4%) proved to be histopathologically advanced (adenocarcinoma, high-grade dysplasia, or villous content), including two cancers and 38 tubulovillous adenomas. Advanced polyps showed mean volume growth of +178% per year (752% per year for adenocarcinomas) compared with +33% per year for nonadvanced polyps and -3% per year for unresected, unretrieved, or resolved polyps (P < .001). In addition, 90% of histologically advanced polyps achieved a volume of 100 mm3 and/or volume growth rate of 100% per year, compared with 29% of nonadvanced and 16% of unresected or resolved polyps (P < .001). Polyp volume-to-diameter ratio was also significantly greater for advanced polyps. For polyps observed at three or more time points, most advanced polyps demonstrated an initial slower growth interval, followed by a period of more rapid growth. Conclusion Small colorectal polyps ultimately proving to be histopathologically advanced neoplasms demonstrated substantially faster growth and attained greater overall size compared with nonadvanced polyps. Clinical trial registration no. NCT00204867 © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Dachman in this issue.

Development of a next-generation sequencing protocol for the canine T cell receptor beta chain repertoire.

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1-2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

NK cells propagate T cell immunity following in situ tumor vaccination.

We report an in situ vaccination, adaptable to nearly any type of cancer, that combines radiotherapy targeting one tumor and intratumoral injection of this site with tumor-specific antibody and interleukin-2 (IL-2; 3xTx). In a phase I clinical trial, administration of 3xTx (with an immunocytokine fusion of tumor-specific antibody and IL-2, hu14.18-IL2) to subjects with metastatic melanoma increases peripheral CD8+ T cell effector polyfunctionality. This suggests the potential for 3xTx to promote antitumor immunity against metastatic tumors. In poorly immunogenic syngeneic murine melanoma or head and neck carcinoma models, 3xTx stimulates CD8+ T cell-mediated antitumor responses at targeted and non-targeted tumors. During 3xTx treatment, natural killer (NK) cells promote CTLA4+ regulatory T cell (Treg) apoptosis in non-targeted tumors. This is dependent on NK cell expression of CD86, which is upregulated downstream of KLRK1. NK cell depletion increases Treg infiltration, diminishing CD8+ T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8+ T cell immunity following 3xTx.

Novel autoantibodies help diagnose anti-SSA antibody negative Sjögren's disease and predict abnormal labial salivary gland pathology.

Objectives: Sj□gren's disease (SjD) diagnosis requires either positive anti-SSA antibodies or a labial salivary gland biopsy with a positive focus score (FS). One-third of SjD patients lack anti-SSA antibodies (SSA-), requiring a positive FS for diagnosis. Our objective was to identify novel autoantibodies to diagnose 'seronegative' SjD. Methods: IgG binding to a high density whole human peptidome array was quantified using sera from SSA- SjD cases and matched non-autoimmune controls. We identified the highest bound peptides using empirical Bayesian statistical filters, which we confirmed in an independent cohort comprising SSA- SjD (n=76), sicca controls without autoimmunity (n=75), and autoimmune controls (SjD features but not meeting SjD criteria; n=41). In this external validation, we used non-parametric methods for peptide abundance and controlled false discovery rate in group comparisons. For predictive modeling, we used logistic regression, model selection methods, and cross-validation to identify clinical and peptide variables that predict SSA- SjD and FS positivity. Results: IgG against a peptide from D-aminoacyl-tRNA deacylase (DTD2) was bound more in SSA- SjD than sicca controls (p=.004) and more than combined controls (sicca and autoimmune controls combined; p=0.003). IgG against peptides from retroelement silencing factor-1 (RESF1) and DTD2, were bound more in FS-positive than FS-negative participants (p=.010; p=0.012). A predictive model incorporating clinical variables showed good discrimination between SjD versus control (AUC 74%) and between FS-positive versus FS-negative (AUC 72%). Conclusion: We present novel autoantibodies in SSA- SjD that have good predictive value for SSA- SjD and FS-positivity. KEY MESSAGES: What is already known on this topic - Seronegative (anti-SSA antibody negative [SSA-]) Sjögren's disease (SjD) requires a labial salivary gland biopsy for diagnosis, which is challenging to obtain and interpret. What this study adds - We identified novel autoantibodies in SSA- SjD that, when combined with readily available clinical variables, provide good predictive ability to discriminate 1) SSA- SjD from control participants and 2) abnormal salivary gland biopsies from normal salivary gland biopsies. How this study might affect research, practice or policy - This study provides novel diagnostic antibodies addressing the critical need for improvement of SSA- SjD diagnostic tools.

SURROGATE SELECTION OVERSAMPLES EXPANDED T CELL CLONOTYPES.

Inference from immunological data on cells in the adaptive immune system may benefit from modeling specifications that describe variation in the sizes of various clonal sub-populations. We develop one such specification in order to quantify the effects of surrogate selection assays, which we confirm may lead to an enrichment for amplified, potentially disease-relevant T cell clones. Our specification couples within-clonotype birth-death processes with an exchangeable model across clonotypes. Beyond enrichment questions about the surrogate selection design, our framework enables a study of sampling properties of elementary sample diversity statistics; it also points to new statistics that may usefully measure the burden of somatic genomic alterations associated with clonal expansion. We examine statistical properties of immunological samples governed by the coupled model specification, and we illustrate calculations in surrogate selection studies of melanoma and in single-cell genomic studies of T cell repertoires.

Growth rates and histopathological outcomes of small (6-9 mm) colorectal polyps based on CT colonography surveillance and endoscopic removal.

BACKGROUND AND AIMS: The natural history of small polyps is not well established and rests on limited evidence from barium enema studies decades ago. Patients with one or two small polyps (6-9 mm) at screening CT colonography (CTC) are offered CTC surveillance at 3 years but may elect immediate colonoscopy. This practice allows direct observation of the growth of subcentimetre polyps, with histopathological correlation in patients undergoing subsequent polypectomy. DESIGN: Of 11 165 asymptomatic patients screened by CTC over a period of 16.4 years, 1067 had one or two 6-9 mm polyps detected (with no polyps ≥10 mm). Of these, 314 (mean age, 57.4 years; M:F, 141:173; 375 total polyps) elected immediate colonoscopic polypectomy, and 382 (mean age 57.0 years; M:F, 217:165; 481 total polyps) elected CTC surveillance over a mean of 4.7 years. Volumetric polyp growth was analysed, with histopathological correlation for resected polyps. Polyp growth and regression were defined as volume change of ±20% per year, with rapid growth defined as +100% per year (annual volume doubling). Regression analysis was performed to evaluate predictors of advanced histology, defined as the presence of cancer, high-grade dysplasia (HGD) or villous components. RESULTS: Of the 314 patients who underwent immediate polypectomy, 67.8% (213/314) harboured adenomas, 2.2% (7/314) with advanced histology; no polyps contained cancer or HGD. Of 382 patients who underwent CTC surveillance, 24.9% (95/382) had polyps that grew, while 62.0% (237/382) remained stable and 13.1% (50/382) regressed in size. Of the 58.6% (224/382) CTC surveillance patients who ultimately underwent colonoscopic resection, 87.1% (195/224) harboured adenomas, 12.9% (29/224) with advanced histology. Of CTC surveillance patients with growing polyps who underwent resection, 23.2% (19/82) harboured advanced histology vs 7.0% (10/142) with stable or regressing polyps (OR: 4.0; p<0.001), with even greater risk of advanced histology in those with rapid growth (63.6%, 14/22, OR: 25.4; p<0.001). Polyp growth, but not patient age/sex or polyp morphology/location were significant predictors of advanced histology. CONCLUSION: Small 6-9 mm polyps present overall low risk to patients, with polyp growth strongly associated with higher risk lesions. Most patients (75%) with small 6-9 mm polyps will see polyp stability or regression, with advanced histology seen in only 7%. The minority of patients (25%) with small polyps that do grow have a 3-fold increased risk of advanced histology.

Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.

Bayes optimal informer sets for early-stage drug discovery.

An important experimental design problem in early-stage drug discovery is how to prioritize available compounds for testing when very little is known about the target protein. Informer-based ranking (IBR) methods address the prioritization problem when the compounds have provided bioactivity data on other potentially relevant targets. An IBR method selects an informer set of compounds, and then prioritizes the remaining compounds on the basis of new bioactivity experiments performed with the informer set on the target. We formalize the problem as a two-stage decision problem and introduce the Bayes Optimal Informer SEt (BOISE) method for its solution. BOISE leverages a flexible model of the initial bioactivity data, a relevant loss function, and effective computational schemes to resolve the two-step design problem. We evaluate BOISE and compare it to other IBR strategies in two retrospective studies, one on protein-kinase inhibition and the other on anticancer drug sensitivity. In both empirical settings BOISE exhibits better predictive performance than available methods. It also behaves well with missing data, where methods that use matrix completion show worse predictive performance.

Role of dual specificity phosphatases (DUSPs) in melanoma cellular plasticity and drug resistance.

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often associated with drug resistance. The mechanisms that control melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that emergence of MAPK inhibitor (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated melanoma cells. Here, we show that knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decrease the survival of MAPKi-resistant cells and sensitizes them to BRAFi and MEKi. Pharmacological inhibition of DUSP1/6 upregulated nestin, a neural crest stem cell marker, in both MAPKi-sensitive cells and cells with acquired MAPKi-resistance. In contrast, treatment with BCI resulted in upregulation of MAP2, a neuronal differentiation marker, only in MAPKi-sensitive cells but caused downregulation of both MAP2 and GFAP, a glial marker, in all MAPKi-resistant cell lines. These data suggest that DUSP proteins are involved in the regulation of cellular plasticity cells and melanoma drug resistance and are potential targets for treatment of MAPKi-resistant melanoma.

Molecular characterization of hypoxanthine guanine phosphoribosyltransferase mutant T cells in human blood: The concept of surrogate selection for immunologically relevant cells.

Somatic cell gene mutations arise in vivo due to replication errors during DNA synthesis occurring spontaneously during normal DNA synthesis or as a result of replication on a DNA template damaged by endogenous or exogenous mutagens. In principle, changes in the frequencies of mutant cells in vivo in humans reflect changes in exposures to exogenous or endogenous DNA damaging insults, other factors being equal. It is becoming increasingly evident however, that somatic mutations in humans have a far greater range of interpretations. For example, mutations in lymphocytes provide invaluable probes for in vivo cellular and molecular processes, providing identification of clonal amplifications of these cells in autoimmune and infectious diseases, transplantation recipients, paroxysmal nocturnal hemoglobinuria (PNH), and cancer. The assay for mutations of the X-chromosomal hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has gained popular acceptance for this purpose since viable mutant cells can be recovered for molecular and other analyses. Although the major application of the HPRT T cell assay remains human population monitoring, the enrichment of activated T cells in the mutant fraction in individuals with ongoing immunological processes has demonstrated the utility of surrogate selection, a method that uses somatic mutation as a surrogate marker for the in vivo T cell proliferation that underlies immunological processes to investigate clinical disorders with immunological features. Studies encompassing a wide range of clinical conditions are reviewed. Despite the historical importance of the HPRT mutation system in validating surrogate selection, there are now additional mutational and other methods for identifying immunologically active T cells. These methods are reviewed and provide insights for strategies to extend surrogate selection in future studies.

SpotClean adjusts for spot swapping in spatial transcriptomics data.

Spatial transcriptomics is a powerful and widely used approach for profiling the gene expression landscape across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind RNA. Ideally, unique molecular identifiers (UMIs) at a spot measure spot-specific expression, but this is often not the case in practice due to bleed from nearby spots, an artifact we refer to as spot swapping. To improve the power and precision of downstream analyses in spatial transcriptomics experiments, we propose SpotClean, a probabilistic model that adjusts for spot swapping to provide more accurate estimates of gene-specific UMI counts. SpotClean provides substantial improvements in marker gene analyses and in clustering, especially when tissue regions are not easily separated. As demonstrated in multiple studies of cancer, SpotClean improves tumor versus normal tissue delineation and improves tumor burden estimation thus increasing the potential for clinical and diagnostic applications of spatial transcriptomics technologies.

A Revised Markov Model Evaluating Oophorectomy at the Time of Hysterectomy for Benign Indication: Age 65 Years Revisited.

OBJECTIVE: To perform an updated Markov modeling to assess the optimal age for bilateral salpingo-oophorectomy (BSO) at the time of hysterectomy for benign indication. METHODS: We performed a literature review that assessed hazard ratios (HRs) for mortality by disease, age, hysterectomy with or without BSO, and estrogen therapy use. Base mortality rates were derived from national vital statistics data. A Markov model from reported HRs predicted the proportion of the population staying alive to age 80 years by 1-year and 5-year age groups at time of surgery, from age 45 to 55 years. Those younger than age 50 years were modeled as either taking postoperative estrogen or not; those 50 and older were modeled as not receiving estrogen. Computations were performed with R 3.5.1, using Bayesian integration for HR uncertainty. RESULTS: Performing salpingo-oophorectomy before age 50 years for those not taking estrogen yields a lower survival proportion to age 80 years than hysterectomy alone before age 50 years (52.8% [Bayesian CI 40.7-59.7] vs 63.5% [Bayesian CI 62.2-64.9]). At or after age 50 years, there were similar proportions of those living to age 80 years with hysterectomy alone (66.4%, Bayesian CI 65.0-67.6) compared with concurrent salpingo-oophorectomy (66.9%, Bayesian CI 64.4-69.0). Importantly, those taking estrogen when salpingo-oophorectomy was performed before age 50 years had similar proportions of cardiovascular disease, stroke, and people living to age 80 years as those undergoing hysterectomy alone or those undergoing hysterectomy and salpingo-oophorectomy at age 50 years and older. CONCLUSION: This updated Markov model argues for the consideration of concurrent salpingo-oophorectomy for patients who are undergoing hysterectomy at age 50 and older and suggests that initiating estrogen in those who need salpingo-oophorectomy before age 50 years mitigates increased mortality risk.

GM-CSF elicits antibodies to tumor-associated proteins when used as a prostate cancer vaccine adjuvant.

Antibody responses to off-target cancer-associated proteins have been detected following immunotherapies for cancer, suggesting these may be the result of antigen spread. We have previously reported that serum antibodies to prostate cancer-associated proteins were detectable using a high-throughput peptide array. We hypothesized that the breadth of antibody responses elicited by a vaccine could serve as a measure of the magnitude of its induced antigen spread. Consequently, sera from patients with prostate cancer, treated prior to or after vaccination in one of four separate clinical trials, were evaluated for antibody responses to an array of 177,604 peptides derived from over 1600 prostate cancer-associated gene products. Antibody responses to the same group of 5680 peptides previously reported were identified following vaccinations in which patients were administered GM-CSF as an adjuvant, but not with vaccine in the absence of GM-CSF. Hence, antibody responses to off-target proteins following vaccination may not necessarily serve as evidence of antigen spread and must be interpreted with particular caution following vaccine strategies that use GM-CSF, as GM-CSF appears to have direct effects on the production of antibodies. The evaluation of T cell responses to non-target antigens is likely a preferred approach for detection of immune-mediated antigen spread.

Rheumatoid Factor and Anti-Modified Protein Antibody Reactivities Converge on IgG Epitopes.

OBJECTIVE: Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG. METHODS: Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA. RESULTS: Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc. CONCLUSION: The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

SpatialCorr identifies gene sets with spatially varying correlation structure.

Recent advances in spatially resolved transcriptomics technologies enable both the measurement of genome-wide gene expression profiles and their mapping to spatial locations within a tissue. A first step in spatial transcriptomics data analysis is identifying genes with expression that varies spatially, and robust statistical methods exist to address this challenge. While useful, these methods do not detect spatial changes in the coordinated expression within a group of genes. To this end, we present SpatialCorr, a method for identifying sets of genes with spatially varying correlation structure. Given a collection of gene sets pre-defined by a user, SpatialCorr tests for spatially induced differences in the correlation of each gene set within tissue regions, as well as between and among regions. An application to cutaneous squamous cell carcinoma demonstrates the power of the approach for revealing biological insights not identified using existing methods.

Dimension constraints improve hypothesis testing for large-scale, graph-associated, brain-image data.

For large-scale testing with graph-associated data, we present an empirical Bayes mixture technique to score local false-discovery rates (FDRs). Compared to procedures that ignore the graph, the proposed Graph-based Mixture Model (GraphMM) method gains power in settings where non-null cases form connected subgraphs, and it does so by regularizing parameter contrasts between testing units. Simulations show that GraphMM controls the FDR in a variety of settings, though it may lose control with excessive regularization. On magnetic resonance imaging data from a study of brain changes associated with the onset of Alzheimer's disease, GraphMM produces greater yield than conventional large-scale testing procedures.

Validation of genetic classifiers derived from mouse and human tumors to identify molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Standard treatment for advanced-stage CRC for decades has included 5-fluorouracil-based chemotherapy. More recently, targeted therapies for metastatic CRC are being used based on the individual cancer's molecular profile. In the past few years, several different molecular subtype schemes for human CRC have been developed. The molecular subtypes can be distinguished by gene expression signatures and have the potential to be used to guide treatment decisions. However, many subtyping classification methods were developed using mRNA expression levels of hundreds to thousands of genes, making them impractical for clinical use. In this study, we assessed whether an immunohistochemical approach could be used for molecular subtyping of CRCs. We validated two previously published, independent sets of immunohistochemistry classifiers and modified the published methods to improve the accuracy of the scoring methods. In addition, we evaluated whether protein and genetic signatures identified originally in the mouse were linked to clinical outcomes of patients with CRC. We found that low DDAH1 or low GAL3ST2 protein levels in human CRCs correlate with poor patient outcomes. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for patients with CRC.

Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

BACKGROUND: Data are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process. AIM: We sought to determine whether tumors with a multi-ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention. METHODS: Mice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red. RESULTS: Cell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi-ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low-dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell. CONCLUSION: These data indicate that genetically distinct tumor-founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi-ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.