A comparison of DNA sequencing and gene expression profiling to assist tissue of origin diagnosis in cancer of unknown primary.

Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype.

BACKGROUND: Autoimmune diseases comprise a spectrum of illnesses and are on the rise worldwide. Although antinuclear antibodies (ANAs) are detected in many autoimmune diseases, up to 20% of healthy women are ANA-positive (ANA+) and most will never develop clinical symptoms. Furthermore, disease transition is higher among ANA+ African Americans compared with ANA+ European Americans. OBJECTIVE: We sought to determine the immune features that might define and prevent transition to clinical autoimmunity in ANA+ healthy individuals. METHODS: We comprehensively phenotyped immune profiles of African Americans and European Americans who are ANA-negative (ANA-) healthy, ANA+ healthy, or have SLE using single cell mass cytometry, next-generation RNA-sequencing, multiplex cytokine profiling, and phospho-signaling analyses. RESULTS: We found that, compared with both ANA- and ANA+ healthy individuals, patients with SLE of both races displayed T-cell expansion and elevated expression of type I and II interferon pathways. We discovered a unique immune signature that suggests a suppressive immune phenotype and reduced CD11C+ autoimmunity-associated B cells in healthy ANA+ European Americans that is absent in their SLE or even healthy ANA- counterparts, or among African American cohorts. In contrast, ANA+ healthy African Americans exhibited elevated expression of T-cell activation markers and higher plasma levels of IL-6 than did healthy ANA+ European Americans. CONCLUSIONS: We propose that this novel immune signature identified in ANA+ healthy European Americans may protect them from T-cell expansion, heightened activation of interferon pathways, and disease transition.

Dysregulation of innate and adaptive serum mediators precedes systemic lupus erythematosus classification and improves prognostic accuracy of autoantibodies.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a poorly understood preclinical stage of immune dysregulation and symptom accrual. Accumulation of antinuclear autoantibody (ANA) specificities is a hallmark of impending clinical disease. Yet, many ANA-positive individuals remain healthy, suggesting that additional immune dysregulation underlies SLE pathogenesis. Indeed, we have recently demonstrated that interferon (IFN) pathways are dysregulated in preclinical SLE. To determine if other forms of immune dysregulation contribute to preclinical SLE pathogenesis, we measured SLE-associated autoantibodies and soluble mediators in samples from 84 individuals collected prior to SLE classification (average timespan = 5.98 years), compared to unaffected, healthy control samples matched by race, gender, age (±5 years), and time of sample procurement. We found that multiple soluble mediators, including interleukin (IL)-5, IL-6, and IFN-γ, were significantly elevated in cases compared to controls more than 3.5 years pre-classification, prior to or concurrent with autoantibody positivity. Additional mediators, including innate cytokines, IFN-associated chemokines, and soluble tumor necrosis factor (TNF) superfamily mediators increased longitudinally in cases approaching SLE classification, but not in controls. In particular, levels of B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) were comparable in cases and controls until less than 10 months pre-classification. Over the entire pre-classification period, random forest models incorporating ANA and anti-Ro/SSA positivity with levels of IL-5, IL-6, and the IFN-γ-induced chemokine, MIG, distinguished future SLE patients with 92% (±1.8%) accuracy, compared to 78% accuracy utilizing ANA positivity alone. These data suggest that immune dysregulation involving multiple pathways contributes to SLE pathogenesis. Importantly, distinct immunological profiles are predictive for individuals who will develop clinical SLE and may be useful for delineating early pathogenesis, discovering therapeutic targets, and designing prevention trials.

Influenza A (H1N1) virus infection triggers severe pulmonary inflammation in lupus-prone mice following viral clearance.

Each year, up to one fifth of the United States population is infected with influenza virus. Although mortality rates are low, hundreds of thousands are hospitalized each year in the United States. Specific high risk groups, such as those with suppressed or dysregulated immune systems, are at greater danger for influenza complications. Respiratory infections are a common cause of hospitalizations and early mortality in patients with systemic lupus erythematosus (SLE); however, whether this increased infection risk is a consequence of the underlying dysregulated immune background and/or immunosuppressing drugs is unknown. To evaluate the influenza immune response in the context of lupus, as well as assess the effect of infection on autoimmune disease in a controlled setting, we infected lupus-prone MRL/MpJ-Fas(lpr) mice with influenza virus A PR/8/34 H1N1. Interestingly, we found that Fas(lpr) mice generated more influenza A virus specific T cells with less neutrophil accumulation in the lung during acute infection. Moreover, Fas(lpr) mice produced fewer flu-specific IgG and IgM antibodies, but effectively cleared the virus. Further, increased extrinsic apoptosis during influenza infection led to a delay in autoimmune disease pathology with decreased severity of splenomegaly and kidney disease. Following primary influenza A infection, Fas(lpr) mice had severe complications during the contraction and resolution phase with widespread severe pulmonary inflammation. Our findings suggest that influenza infection may not exacerbate autoimmune pathology in mice during acute infection as a direct result of virus induced apoptosis. Additionally, autoimmunity drives an enhanced antigen-specific T cell response to clear the virus, but persisting pulmonary inflammation following viral clearance may cause complications in this lupus animal model.

Stroke with intracranial stenosis is associated with increased platelet activation in sickle cell anemia.

BACKGROUND: Overt stroke in sickle cell anemia (SCA) is associated with intracranial stenosis and thrombus formation. Platelet activation is critical for thrombus formation. PROCEDURE: Platelet activation studies were performed in 50 subjects: 18 SCA patients with history of stroke or abnormal transcranial Doppler (TCD) and intracranial stenosis seen by magnetic resonance angiogram (MRA), 7 SCA patients with history of stroke or abnormal TCD but no intracranial stenosis, 13 SCA patients with no history of stroke or abnormal TCD, and 12 healthy African-Americans. RESULTS: Of the 18 patients with intracranial stenosis, 11 (61%) had evidence of the moyo-moya phenomenon on MRA. SCA children with intracranial stenosis had a significantly greater total white cell count compared to both healthy African-American controls and SCA patients in the steady-state (P < 0.001). In addition, SCA patients with history of stroke or abnormal TCD had a significantly higher platelet count compared to healthy African-American controls (P < 0.002). The percentage of platelet surface P-selectin expression was significantly greater in patients with intracranial stenosis compared to the other groups (P < 0.05), particularly in individuals that did not have the moya-moya phenomenon seen on MRA. CONCLUSION: Stroke with intracranial stenosis is associated with increased platelet activation in sickle cell anemia, and further investigation is needed on the role of anti-platelet agents in this high-risk population.

New extralimital breeding records of saltmarsh sparrows (Ammospiza caudacuta) and Nelson's sparrows (Ammospiza nelsoni) and their implications.

Saltmarsh (Ammospiza caudacuta) and Nelson's (A. nelsoni) sparrows are sister taxa that breed in tidal marshes along the coast of the Northeastern United States and Canada. The Saltmarsh Sparrow breeds from mid-coast Maine south to Virginia, while the Acadian Nelson's Sparrow breeds from the Canadian maritime provinces south to northern Massachusetts. Here, we present three extralimital observations of breeding Saltmarsh (n = 2) and Nelson's (n = 1) sparrows. In 2021 and 2022, we observed Saltmarsh Sparrow females attending nests at Mendall Marsh, ME, and Milbridge, ME, respectively, approximately 60 and 110 km beyond the documented northern extent of the Saltmarsh Sparrow breeding range. In 2022, we observed a breeding-condition male Nelson's sparrow singing in the upriver portion of a marsh on Cape Cod, Massachusetts, approximately 115 km beyond the previously documented southern extent of the Nelson's Sparrow breeding range. We confirmed morphological species identification using a panel of microsatellite DNA loci. Due to both the well-documented population declines of these species in the region and the intensity of sampling effort undertaken in recent years, we suggest that these observations likely are not indicative of range expansion. However, they do indicate that these 2 taxa have the capacity to use and successfully reproduce in marshes well beyond their established breeding limits. Our findings provide novel insight into the potential for these taxa to occur and successfully breed outside their documented breeding ranges. Given increased interest in their conservation, these results support the idea that management actions aimed at creating or maintaining nesting habitat across both species ranges could benefit both taxa.

Immune and genomic biomarkers of immunotherapy response in cancer of unknown primary.

BACKGROUND: Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers where a primary tissue of origin (TOO) is uncertain. Most patients with CUP have limited treatment options and poor survival outcomes. Immune checkpoint inhibitors (ICIs) can be efficacious in some patients with CUP, but the optimal predictive biomarkers are unknown. We therefore assessed immune and genomic biomarkers as well as predicted TOO in patients with CUP, including a subset treated with ICIs. METHODS: Patients with CUP were subject to gene-expression profiling (GEP) and DNA panel sequencing. Immune and stromal-related gene expression was explored by NanoString, including genes associated with immunotherapy response (IR) in other solid malignancies. ICI responsive cancer types were assigned based on Food and Drug Administration-approved indications, and either detection of a latent primary tumor or the TOO was suspected based on genomics informed pathology review. Tumor mutation burden (TMB) and gene mutations were also assessed. RESULTS: A total of 219 patients with CUP were included, 215 assessed for TOO in a previous study, with the majority (163) receiving both RNA and DNA tests. Of GEP profiled cases, 33% (59/175) had a high IR gene-expression score. Of the DNA sequenced cases, 16% (32/203) had high TMB (>10 mutations/Mb), including two with mismatch repair deficiency. Low correlation was observed between TMB and an IR score (R=0.26, p<0.001). Among 110 CUPs with a latent primary or suspected TOO, 47% (52/110) belonged to ICI-responsive cancer types. More than half of the CUPs had at least one feature that may predict ICI response (high IR score, high TMB, ICI-responsive cancer type). Among patients with CUP treated with ICIs, 8/28 (29%) responded (2 complete responses and 6 partial responses). Among non-responders, 9 had stable and 11 had progressive disease. All responders had a high IR score (7/8) and/or high TMB (3/8), while most (5/8) belonged to ICI-responsive cancer types. These features were detected at a lower frequency in non-responders and mostly in patients with stable disease. CONCLUSIONS: A significant fraction of CUP tumors had genomic features previously associated with ICI response. High IR score was the most sensitive predictive feature of ICI response, warranting evaluation in a larger patient series.

Unique Serum Immune Phenotypes and Stratification of Oklahoma Native American Rheumatic Disease Patients.

OBJECTIVE: Native American (NA) populations have higher rates of rheumatic disease and present with overlapping disease symptoms and nontraditional serologic features, thus presenting an urgent need for better biomarkers in NA diagnostics. This study used a machine learning approach to identify immune signatures that more effectively stratify NA patients with rheumatic disease. METHODS: Adult NA patients with autoantibody-positive (AAB+) rheumatoid arthritis (RA; n = 28), autoantibody negative (AAB-) RA (n = 18), systemic autoimmune rheumatic disease (n = 28), arthralgia/osteoarthritis (n = 28), or polyarthritis/undifferentiated connective tissue disease (n = 28), and control patients (n = 28) provided serum samples for cytokine, chemokine, and AAB assessment. Random forest clustering and soluble mediator groups were used to identify patients and control patients with similar biologic signatures. The American College of Rheumatology criteria specific for systemic disease and RA identified differences in disease manifestations across clusters. RESULTS: Serum soluble mediators were not homogenous between different NA rheumatic disease diagnostic groups, reflecting the heterogeneity of autoimmune diseases. Clustering by serum biomarkers created 5 analogous immune phenotypes. Soluble mediators and pathways associated with chronic inflammation and involvement of the innate, B cell, T follicular helper cell, and interferon-associated pathways, along with regulatory signatures, distinguished the 5 immune signatures among patients. Select clinical features were associated with individual immune profiles. Patients with low inflammatory and higher regulatory signatures were more likely to have few clinical manifestations, whereas those with T cell pathway involvement had more arthritis. CONCLUSION: Serum protein signatures distinguished NA patients with rheumatic disease into distinct immune subsets. Following these immune profiles over time may assist with earlier diagnoses and help guide more personalized treatment approaches.

Ancestry-based differences in the immune phenotype are associated with lupus activity.

Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.

VESPA: software to facilitate genomic annotation of prokaryotic organisms through integration of proteomic and transcriptomic data.

BACKGROUND: The procedural aspects of genome sequencing and assembly have become relatively inexpensive, yet the full, accurate structural annotation of these genomes remains a challenge. Next-generation sequencing transcriptomics (RNA-Seq), global microarrays, and tandem mass spectrometry (MS/MS)-based proteomics have demonstrated immense value to genome curators as individual sources of information, however, integrating these data types to validate and improve structural annotation remains a major challenge. Current visual and statistical analytic tools are focused on a single data type, or existing software tools are retrofitted to analyze new data forms. We present Visual Exploration and Statistics to Promote Annotation (VESPA) is a new interactive visual analysis software tool focused on assisting scientists with the annotation of prokaryotic genomes though the integration of proteomics and transcriptomics data with current genome location coordinates. RESULTS: VESPA is a desktop Java™ application that integrates high-throughput proteomics data (peptide-centric) and transcriptomics (probe or RNA-Seq) data into a genomic context, all of which can be visualized at three levels of genomic resolution. Data is interrogated via searches linked to the genome visualizations to find regions with high likelihood of mis-annotation. Search results are linked to exports for further validation outside of VESPA or potential coding-regions can be analyzed concurrently with the software through interaction with BLAST. VESPA is demonstrated on two use cases (Yersinia pestis Pestoides F and Synechococcus sp. PCC 7002) to demonstrate the rapid manner in which mis-annotations can be found and explored in VESPA using either proteomics data alone, or in combination with transcriptomic data. CONCLUSIONS: VESPA is an interactive visual analytics tool that integrates high-throughput data into a genomic context to facilitate the discovery of structural mis-annotations in prokaryotic genomes. Data is evaluated via visual analysis across multiple levels of genomic resolution, linked searches and interaction with existing bioinformatics tools. We highlight the novel functionality of VESPA and core programming requirements for visualization of these large heterogeneous datasets for a client-side application. The software is freely available at https://www.biopilot.org/docs/Software/Vespa.php.

Shared and unique immune alterations in pre-clinical autoimmunity.

Progression from health to a classified autoimmune disease is an evolving process that can happen rapidly in some diseases, but usually takes years to develop. Specific immune alterations predate pathogenic autoimmunity and can be used as disease biomarkers to identify high-risk individuals for prevention studies applied in the pre-clinical state. Here we discuss recent findings that illuminate specific immune pathways that are altered in the earliest phases of pre-clinical autoimmunity as well as those mediators more closely associated with later clinically apparent and classified disease onset.

Mycophenolate mofetil reduces STAT3 phosphorylation in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is a highly variable autoimmune disease that can involve severe organ-threatening symptoms, such as lupus nephritis. Certain drugs, such as mycophenolate mofetil (MMF), are effective at reducing morbidity associated with nephritis; however, the immune pathways associated with disease suppression are poorly defined. Here, we provide evidence that MMF inhibits phosphorylation of STAT3 and other associated immune pathways. Using mass cytometry and bead-based or ELISA assays, the systemic phenotype of SLE patients not taking (MMF-) or taking (MMF+) MMF were studied. MMF+ SLE patients had significant reductions in total numbers of transitional B cells, plasmablasts, and T cells, specifically CD4+ Th17-type and CD4+ Treg-type cells, compared with MMF- patients. Plasma soluble mediators were decreased in MMF+ patients including chemokines (MIG/CXCL9 and SDF-1α/CXCL12) and growth factors (VEGF-A and PDGF-BB). Soluble mediators and cell subsets grouped by functional properties revealed significant modifications associated with STAT3 and B cell pathways. Further, healthy PBMCs treated with IL-6 revealed a reduction in p-STAT3 following the addition of mycophenolic acid (the active metabolite of MMF). In conclusion, the inhibition of STAT3 phosphorylation by MMF may explain the effectiveness of this treatment in SLE patients, since increased levels of p-STAT3 are associated with disease pathology.

Autoantibody-Positive Healthy Individuals Display Unique Immune Profiles That May Regulate Autoimmunity.

OBJECTIVE: Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5-8%. Immunologic differences between ANA-positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA-positive individuals avoid transition to clinical autoimmune disease. METHODS: Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjögren's syndrome. From this screening, 31 European American ANA-positive healthy individuals were selected and demographically matched to ANA-negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry. RESULTS: Of 790 individuals screened, 57 (7%) were ANA-positive. The majority of proinflammatory cytokines, including interferon-γ (IFNγ), tumor necrosis factor, interleukin-17 (IL-17), and granulocyte colony-stimulating factor, exhibited a stepwise increase in serum levels from ANA-negative controls to ANA-positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNß, IL-12p40, and stem cell factor/c-Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA-positive individuals (P < 0.001). Further, IL-1 receptor antagonist (IL-1Ra) was down-regulated in SLE patients only (P < 0.0001). ANA-positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT-1 and pSTAT-3 following IFNα stimulation compared with ANA-negative controls (P < 0.05). CONCLUSION: ANA-positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL-12p40, and stem cell factor/c-Kit ligand. Further, severely decreased levels of IL-1Ra in SLE patients compared with ANA-positive individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis.