Immunosignature system for diagnosis of cancer

Although the search for disease biomarkers continues, the clinical return has thus far been disappointing. The complexity of the body’s response to disease makes it difficult to represent this response with only a few biomarkers, particularly when many are present at low levels. An alternative to the typical reductionist biomarker paradigm is an assay we call an “immunosignature.” This approach leverages the response of antibodies to disease-related changes, as well as the inherent signal amplification associated with antigen-stimulated B-cell proliferation. To perform an immunosignature assay, the antibodies in diluted blood are incubated with a microarray of thousands of random sequence peptides. The pattern of binding to these peptides is the immunosignature. Because the peptide sequences are completely random, the assay is effectively disease-agnostic, potentially providing a comprehensive diagnostic on multiple diseases simultaneously. To explore the ability of an immunosignature to detect and identify multiple diseases simultaneously, 20 samples from each of five cancer cohorts collected from multiple sites and 20 noncancer samples (120 total) were used as a training set to develop a reference immunosignature. A blinded evaluation of 120 blinded samples covering the same diseases gave 95% classification accuracy. To investigate the breadth of the approach and test sensitivity to biological diversity further, immunosignatures of >1,500 historical samples comprising 14 different diseases were examined by training with 75% of the samples and testing the remaining 25%. The average accuracy was >98%. These results demonstrate the potential power of the immunosignature approach in the accurate, simultaneous classification of disease.Cancer is the most likely disease for which an early diagnostic would be immediately beneficial. Unfortunately, finding specific biomarkers, especially for cancer, has been complicated by the fact that biological molecules (RNA, DNA, proteins, or peptides) that are uniquely released by a small tumor into the bloodstream are extremely dilute. Classical biomarker assays are based on one-to-one molecular recognition events to detect one or a few specific analytes that are often measured by antibody–protein interactions. There are three fundamental limitations with this approach, all of which are confounded by the dilution problem alluded to above. The first is that the cross-reactivity of such interactions poses a formidable problem in distinguishing diseases. Biology’s promiscuous use of a limited number of homologous sequences, folds, and domains makes specificity difficult. The second is that diseases such as cancer are themselves heterogeneous, and individual response to disease, at a molecular level, can vary considerably. It is unlikely that this level of complexity can be quantitatively assessed by one or a few specific proteins or metabolites in a way that supports robust diagnosis. Third, many of the biomarkers that have been proposed are of low stability or require substantial preassay purification or preparation; these aspects introduce substantial variation into the measured values (1, 2). As a result, although considerable effort has been put into the development of biomarkers, only a small fraction of candidates make it to clinical practice, and the utility of those that are used is sometimes only modest (3–5). Here, we explore the ability of the immunosignature technology to address the ideal of a simple, comprehensive diagnostic for multiple cancers.An “immunosignature” is the pattern obtained when circulating antibodies in blood are allowed to bind to a large microarray of randomized-sequence peptides affixed to a solid surface (6). Cancers generate neoantigens by virtue of their mutagenic nature, and they tend to release native proteins and biomolecules not normally encountered by the immune system (7–9). These behaviors can elicit an immune response (6, 10, 11). By virtue of the tremendous amplification afforded by B-cell replication (12), the signal elicited by the disease-specific antigens is massively amplified. In fact, a key aspect of the immunosignature assay is that the blood is greatly diluted before application to the array, such that only the antibodies that have been sufficiently amplified give distinct signals (13).Another somewhat counterintuitive aspect of the method is that the peptide sequences used on the microarray are purposefully not chosen to represent the natural antigens of the antibodies produced in response to disease. In fact, in the arrays of 10,000 peptides used in this study, the peptide sequences were generated with a random number generator. This enables the same microarray to be used for diagnosis of any disease. Despite using random-sequence peptides, monoclonal antibodies generated from a wide variety of antigens show specific patterns of binding on these arrays, to both cognate and noncognate sequences (14, 15). Many of the peptides bound by a monoclonal antibody against a known linear epitope have no obvious sequence similarity to that epitope. Most of the peptides thus identified have demonstrated low affinity in solution for the antibody but are retained on the arrays due to avidity created by close spacing of individual peptides (15).An immunosignature of an individual consists of an overlay of the patterns from the binding signals of many of the most prominent circulating antibodies. Some of the binding signals are present in most individuals (whether sick or healthy), and some are unique to an individual, but if the individual has a disease such as a cancer, a subset of the binding signals will be due to disease-associated antigens that are common to most individuals with the disease (16). An important aspect of this approach is that it senses essentially all antibodies raised to the disease and detects each of the antibodies as separable binding patterns composed of unique molecular recognition elements. This differs from, for example, an ELISA, which might sum the contributions of many different antibodies using a single protein, cell, or virus capsid. Again, from a statistical perspective, the high dimensionality of this readout affords much more specificity than could be obtained from a set of cognate sequences or from an array of the native antigens themselves.Not only does the use of highly dilute blood and random peptide sequences in the immunosignature assay paradoxically give rise to improved sensitivity and specificity but these aspects of the assay also result in several other unique benefits of the immunosignature approach. Because of the dilution (1:500 in these studies), blood proteins other than antibodies do not significantly bind to the arrays, meaning that there is no sample preparation involved other than dilution (17). The dilution ensures the assay is sample-sparing. Finally, the assay is disease-agnostic. The arrays can be used for the simultaneous detection and identification of multiple diseases.It is simultaneous detection and identification of multiple diseases with a single assay that underlies the true potential of this approach as a disruptive force in healthcare. This, combined with the fact that serum antibodies are robust to handling (17, 18) such that a drop of blood can be sent dried on filter paper through the mail (17), should enable frequent, inexpensive monitoring for many different diseases. The goal of the current work is to test the multidisease aspect of immunosignatures rigorously. Although the approach has previously been used to discriminate various subtypes of brain cancer (19), it has not yet demonstrated multiplexed cancer diagnosis. Here, we perform a blinded train/test validation study wherein a group of 120 individuals with five different cancers from various geographic regions was used as a training set to define a multicancer signature. The signature predicted the disease status of a test cohort of equal size and composition. To explore the ability of the approach to discriminate between an even larger set of diseases, 1,516 different individuals spanning 14 different disease cohorts plus a diverse cohort of healthy controls were assayed and the ability to distinguish between these diseases was evaluated.     

Factors influencing long‐term outcome after kidney transplantation

Many factors influence the long‐term outcome of kidney transplantation, which is defined very schematically by patient death or renal dysfunction leading to graft loss. The most important of these factors is most likely the quality of the transplant itself, with kidneys from living donors showing a positive impact, while kidneys from expanded criteria donors show deleterious impacts. Various clinicopathological scores exist to predict mid‐ to long‐term outcomes and avoid the transplantation of kidneys displaying inferior results. The key factors related to the recipient include their age as well as disease recurrence, HLA matching, HLA immunization, ethnic background, time on dialysis, and cardiovascular comorbidities. Renal function, defined based on estimated GFR and/or proteinuria values, is a result of all these factors. Delayed graft function has a detrimental long‐term impact, as does the level of renal function impairment either in stable condition or in case of progressing dysfunction. Finally, although current immunosuppression regimes are highly efficient in preventing acute rejection, the burden of specific (diabetes, nephrotoxicity) and nonspecific (infection and cancer) side effects has significant negative long‐term consequences that may well be worse in the future because of the increasing ages of both donors and recipients. The development of safer immunosuppression strategies is therefore crucial to improve long‐term outcomes.     

Retrospective chart review of severe infusion reactions with rituximab,cetuximab, and bevacizumab in community oncology practices: assessment of clinical consequences

GOALS OF WORK: Monoclonal antibody (MoAb) treatments can result in severe infusion reactions. Managing infusion reactions in the outpatient setting introduces clinical and resource challenges for patients and providers, but there is little information regarding prevention, management, or outcomes of severe infusion reactions. This study represents one of the first attempts to describe the clinical consequences of severe infusion reactions associated with MoAb treatment. MATERIALS AND METHODS: Clinic staff identified adults treated with rituximab, cetuximab, or bevacizumab who experienced a grade 3 or higher (severe) infusion reaction. Chart reviews from 19 oncology practice sites across the USA captured patient demographics, infusion reaction management procedures, and clinical outcomes. MAIN RESULTS: With an average age of 62 years, the sample comprised of 76 patients who experienced a severe infusion reaction while receiving rituximab (n = 47), cetuximab (n = 24), and bevacizumab (n = 5). The most common pretreatment medications were acetaminophen and antihistamine in the rituximab group and corticosteroids (42%) in the cetuximab group. All cetuximab and the majority of rituximab severe infusion reactions occurred during the first cycle of therapy. Postinfusion reaction management typically included corticosteroids, oxygen, and intravenous fluids. Overall, 22% were hospitalized for a mean of 4 days (range = 2.0 to 6.0 days). Permanent discontinuation of MoAb therapy occurred after the majority of cetuximab (79 to 100%) related severe infusion reactions. CONCLUSIONS: Severe infusion reactions are intensive events that present a serious challenge to patients and oncology practices. Efforts to prevent or reduce such reactions could be of great benefit.     

The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (V_H) region locus was determined and 43 novel V_H segments were identified. The region contains 123 V_H segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 V_H segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of V_H region was calculated to be ∼6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional V_H segments than in pseudogenes. Phylogenetic analysis of 114 V_H segments clearly showed clustering of the V_H segments of each family. However, an independent branch in the tree contained a single V_H, V4-44.1P, sharing similar levels of homology to human V_H families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.     

Antibody‐mediated rejection in pediatric small bowel transplantation: Capillaritis is a major determinant of C4d positivity in intestinal transplant biopsies

The diagnostic criteria for antibody‐mediated rejection (ABMR) after small bowel transplantation (SBT) are not clearly defined, although the presence of donor‐specific antibodies (DSAs) has been reported to be deleterious for graft survival. We aimed to determine the incidence and prognostic value of DSAs and C4d in pediatric SBT and to identify the histopathologic features associated with C4d positivity. We studied all intestinal biopsies (IBx) obtained in the first year posttransplantation (N = 345) in a prospective cohort of 23 children. DSAs and their capacity to fix C1q were identified by using Luminex technology. Eighteen patients (78%) had DSAs, and 9 had the capacity to fix C1q. Seventy‐eight IBx (22.6%) were C4d positive. The independent determinants of C4d positivity were capillaritis grades 2 and 3 (odds ratio [OR] 4.02, P = .047 and OR 5.17, P = .003, respectively), mucosal erosion/ulceration (OR 2.8, P = .019), lamina propria inflammation grades 1 and 2/3 (OR 1.95, P = .043 and OR 3.1, P = .016, respectively), and chorion edema (OR 2.16, P = .028). Complement‐fixing DSAs and repeated C4d‐positive IBx were associated with poor outcome (P = .021 and P = .001, respectively). Our results support that capillaritis should be considered as a feature of ABMR in SBT and identify C1q‐fixing DSAs and repeated C4d positivity as potential markers of poor outcome.     

Human leukocyte antigens antibodies after lung transplantation: Primary results of the HALT study

Donor‐specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) are associated with worse outcomes after lung transplantation. To determine the incidence and characteristics of DSA early after lung transplantation, we conducted a prospective multicenter observational study that used standardized treatment and testing protocols. Among 119 transplant recipients, 43 (36%) developed DSA: 6 (14%) developed DSA only to class I HLA, 23 (53%) developed DSA only to class II HLA, and 14 (33%) developed DSA to both class I and class II HLA. The median DSA mean fluorescence intensity (MFI) was 3197. We identified a significant association between the Lung Allocation Score and the development of DSA (HR = 1.02, 95% CI: 1.001‐1.03, P = .047) and a significant association between DSA with an MFI ≥ 3000 and acute cellular rejection (ACR) grade ≥ A2 (HR = 2.11, 95% CI: 1.04‐4.27, P = .039). However, we did not detect an association between DSA and survival. We conclude that DSA occur frequently early after lung transplantation, and most target class II HLA. DSA with an MFI ≥ 3000 have a significant association with ACR. Extended follow‐up is necessary to determine the impact of DSA on other important outcomes.