Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

The Cholangiocarcinoma in the Young (CITY) Study: Tumor Biology, Treatment Patterns, and Survival Outcomes in Adolescent Young Adults With Cholangiocarcinoma.

PURPOSE: Increased awareness of the distinct tumor biology for adolescents and young adults (AYAs) with cancer has led to improvement in outcomes for this population. However, in cholangiocarcinoma (CCA), a paucity of data exist on the AYA population. To our knowledge, we present the largest study to date on AYA disease biology, treatment patterns, and survival outcomes in CCA. METHODS: A multi-institutional cohort of patients with CCA diagnosed with intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma (ECC) was used for analysis. Retrospective chart review was conducted on patients who were 50 years old and younger (young; n = 124) and older than 50 years (older; n = 723). RESULTS: Among 1,039 patients screened, 847 patients met eligibility (72% ICC, 28% ECC). Young patients had a larger median tumor size at resection compared with older patients (4.2 v 3.6 cm; P = .048), more commonly had N1 disease (65% v 43%; P = .040), and were more likely to receive adjuvant therapy (odds ratio, 4.0; 95% CI, 1.64 to 9.74). Tumors of young patients were more likely to harbor an FGFR2 fusion, BRAF mutation, or ATM mutation (P < .05 for each). Young patients were more likely to receive palliative systemic therapy (96% v 69%; P < .001), targeted therapy (23% v 8%; P < .001), and treatment on a clinical trial (31% v 19%; P = .004). Among patients who presented with advanced disease, young patients had a higher median overall survival compared with their older counterparts (17.7 v 13.5 months; 95% CI, 12.6 to 22.6 v 11.4 to 14.8; P = .049). CONCLUSION: Young patients with CCA had more advanced disease at resection, more commonly received both adjuvant and palliative therapies, and demonstrated improved survival compared with older patients. Given the low clinical trial enrollment and poor outcomes among some AYA cancer populations, data to the contrary in CCA are highly encouraging.

Molecular profiling and treatment pattern differences between intrahepatic and extrahepatic cholangiocarcinoma.

BACKGROUND: Treatment patterns for intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) differ, but limited studies exist comparing them. This study examines differences in molecular profiling rates and treatment patterns in these populations, focusing on use of adjuvant, liver-directed, targeted, and investigational therapies. METHODS: This multicenter collaboration included patients with ICC or ECC treated at 1 of 8 participating institutions. Retrospective data were collected on risk factors, pathology, treatments, and survival. Comparative statistical tests were 2-sided. RESULTS: Among 1039 patients screened, 847 patients met eligibility (ICC = 611, ECC = 236). Patients with ECC were more likely than those with ICC to present with early stage disease (53.8% vs 28.0%), undergo surgical resection (55.1% vs 29.8%), and receive adjuvant chemoradiation (36.5% vs 4.2%) (all P < .00001). However, they were less likely to undergo molecular profiling (50.3% vs 64.3%) or receive liver-directed therapy (17.9% vs 35.7%), targeted therapy (4.7% vs 18.9%), and clinical trial therapy (10.6% vs 24.8%) (all P < .001). In patients with recurrent ECC after surgery, the molecular profiling rate was 64.5%. Patients with advanced ECC had a shorter median overall survival than those with advanced ICC (11.8 vs 15.1 months; P < .001). CONCLUSIONS: Patients with advanced ECC have low rates of molecular profiling, possibly in part because of insufficient tissue. They also have low rates of targeted therapy use and clinical trial enrollment. While these rates are higher in advanced ICC, the prognosis for both subtypes of cholangiocarcinoma remains poor, and a pressing need exists for new effective targeted therapies and broader access to clinical trials.

AncesTree: An interactive immunoglobulin lineage tree visualizer.

High-throughput sequencing of human immunoglobulin genes allows analysis of antibody repertoires and the reconstruction of clonal lineage evolution. The study of antibodies (Abs) affinity maturation is of specific interest to understand the generation of Abs with high affinity or broadly neutralizing activities. Moreover, phylogenic analysis enables the identification of the key somatic mutations required to achieve optimal antigen binding. The Immcantation framework provides a start-to-finish set of analytical methods for high-throughput adaptive immune receptor repertoire sequencing (AIRR-Seq; Rep-Seq) data. Furthermore, Immcantation's Change-O package has developed IgPhyML, an algorithm designed to build specifically immunoglobulin (Ig) phylogenic trees. Meanwhile Phylip, an algorithm that has been originally developed for applications in ecology and macroevolution, can also be used for the phylogenic reconstruction of antibodies maturation pathway. To complement Ig lineages made by IgPhyML or Dnaml (Phylip), we developed AncesTree, a graphic user interface (GUI) that aims to give researchers the opportunity to interactively explore antibodies clonal evolution. AncesTree displays interactive immunoglobulins phylogenic tree, Ig related mutations and sequence alignments using additional information coming from specialized antibody tools. The GUI is a Java standalone application allowing interaction with Ig tree that can run under Windows, Linux and Mac OS.

Cosmetic treatment in patients with autoimmune connective tissue diseases: Best practices for patients with lupus erythematosus.

The cutaneous manifestations of lupus, especially chronic cutaneous lupus erythematosus, are a source of significant morbidity and can negatively impact patient quality of life. While the active inflammatory component of the disease may be adequately treated, patients are frequently left with residual skin damage and disfiguring aesthetic deficits. Dermatologists lack guidelines regarding the use and safety of various reconstructive and cosmetic interventions in this patient population. Laser treatments are largely avoided in the lupus population because of the possible photodamaging effects of ultraviolet and visible light. Similarly, given the autoimmune nature of this disease, some physicians avoid injectable treatment and grafts because of the concern for disease reactivation via antigenic stimulation. In the second article in this continuing medical education series we compile available data on this topic with the goal of providing evidence-based guidance on the cosmetic treatment of patients with lupus erythematosus with a focus on chronic cutaneous lupus erythematosus.

Cosmetic treatment in patients with autoimmune connective tissue diseases: Best practices for patients with morphea/systemic sclerosis.

Morphea and systemic sclerosis are inflammatory, sclerosing disorders. Morphea primarily affects the dermis and subcutaneous fat, while systemic sclerosis typically involves the skin and internal organs. Functional impairment and cosmetic disfigurement are common in both diseases. Treatment options to mitigate disease progression remain limited. Both functional impairment and cosmetic deficits negatively impact quality of life and psychological well-being in this patient population. While the number of cosmetic procedures performed in the United States continues to rise each year, limited data exist regarding best practices for correcting aesthetic deficits caused by autoimmune conditions. There is scarce information to guide safety decisions regarding laser parameters, soft tissue augmentation, treatment intervals, and the concurrent use of immune-modifying or immune-suppressing medications. Given the fears of disease reactivation and exacerbation from postprocedural inflammation along with limited data, it is difficult for clinicians to provide evidence-based cosmetic treatment with realistic expectations with regard to short- and long-term outcomes. In the first article in this continuing medical education series, we attempt to address this practice gap.

Persistent Antibody Clonotypes Dominate the Serum Response to Influenza over Multiple Years and Repeated Vaccinations.

Humans are repeatedly exposed to influenza virus via infections and vaccinations. Understanding how multiple exposures and pre-existing immunity impact antibody responses is essential for vaccine development. Given the recent prevalence of influenza H1N1 A/California/7/2009 (CA09), we examined the clonal composition and dynamics of CA09 hemagglutinin (HA)-reactive IgG repertoire over 5 years in a donor with multiple influenza exposures. The anti-CA09 HA polyclonal response in this donor comprised 24 persistent antibody clonotypes, accounting for 72.6% ± 10.0% of the anti-CA09 HA repertoire over 5 years. These persistent antibodies displayed higher somatic hypermutation relative to transient serum antibodies detected at one time point. Additionally, persistent antibodies predominantly demonstrated cross-reactivity and potent neutralization toward a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a feature correlated with HA stem recognition. This analysis reveals how "serological imprinting" impacts responses to influenza and suggests that once elicited, cross-reactive antibodies targeting the HA stem can persist for years.

CDKN2A/CDK4 Status in Greek Patients with Familial Melanoma and Association with Clinico-epidemiological Parameters.

Approximately 5-10% of melanoma cases occur in a familial context. CDKN2A/CDK4 were the first high-penetrance melanoma genes identified. The aims of this study were to evaluate CDKN2A/CDK4 variants in Greek familial melanoma patients and to correlate the mutational status with specific clinico-epidemiological characteristics. A cross-sectional study was conducted by genotyping CDKN2A/CDK4 variants and selected MC1R polymorphisms in 52 melanoma-prone families. Descriptive statistics were calculated and comparisons were made using the χ2 test, Fisher's exact test and Student's t-test for statistical analysis, as appropriate. CDKN2A variants were detected in 46.2% of melanoma-prone families, while a CDK4 variant was found in only one family. This study confirmed that, in the Greek population, the age at melanoma diagnosis was lower in patients carrying a variant in CDKN2A compared with wild-type patients. No statistically significant associations were found between CDKN2A mutational status and MC1R polymorphisms.

Rational development of a protective P. vivax vaccine evaluated with transgenic rodent parasite challenge models.

Development of a protective and broadly-acting vaccine against the most widely distributed human malaria parasite, Plasmodium vivax, will be a major step towards malaria elimination. However, a P. vivax vaccine has remained elusive by the scarcity of pre-clinical models to test protective efficacy and support further clinical trials. In this study, we report the development of a highly protective CSP-based P. vivax vaccine, a virus-like particle (VLP) known as Rv21, able to provide 100% sterile protection against a stringent sporozoite challenge in rodent models to malaria, where IgG2a antibodies were associated with protection in absence of detectable PvCSP-specific T cell responses. Additionally, we generated two novel transgenic rodent P. berghei parasite lines, where the P. berghei csp gene coding sequence has been replaced with either full-length P. vivax VK210 or the allelic VK247 csp that additionally express GFP-Luciferase. Efficacy of Rv21 surpassed viral-vectored vaccination using ChAd63 and MVA. We show for the first time that a chimeric VK210/247 antigen can elicit high level cross-protection against parasites expressing either CSP allele, which provide accessible and affordable models suitable to support the development of P. vivax vaccines candidates. Rv21 is progressing to GMP production and has entered a path towards clinical evaluation.

Rapid development of broadly influenza neutralizing antibodies through redundant mutations.

The neutralizing antibody response to influenza virus is dominated by antibodies that bind to the globular head of haemagglutinin, which undergoes a continuous antigenic drift, necessitating the re-formulation of influenza vaccines on an annual basis. Recently, several laboratories have described a new class of rare influenza-neutralizing antibodies that target a conserved site in the haemagglutinin stem. Most of these antibodies use the heavy-chain variable region VH1-69 gene, and structural data demonstrate that they bind to the haemagglutinin stem through conserved heavy-chain complementarity determining region (HCDR) residues. However, the VH1-69 antibodies are highly mutated and are produced by some but not all individuals, suggesting that several somatic mutations may be required for their development. To address this, here we characterize 197 anti-stem antibodies from a single donor, reconstruct the developmental pathways of several VH1-69 clones and identify two key elements that are required for the initial development of most VH1-69 antibodies: a polymorphic germline-encoded phenylalanine at position 54 and a conserved tyrosine at position 98 in HCDR3. Strikingly, in most cases a single proline to alanine mutation at position 52a in HCDR2 is sufficient to confer high affinity binding to the selecting H1 antigen, consistent with rapid affinity maturation. Surprisingly, additional favourable mutations continue to accumulate, increasing the breadth of reactivity and making both the initial mutations and phenylalanine at position 54 functionally redundant. These results define VH1-69 allele polymorphism, rearrangement of the VDJ gene segments and single somatic mutations as the three requirements for generating broadly neutralizing VH1-69 antibodies and reveal an unexpected redundancy in the affinity maturation process.

Fibroblast growth factor receptor 1-transformed mammary epithelial cells are dependent on RSK activity for growth and survival.

Fibroblast growth factor receptor 1 (FGFR1) is frequently amplified and highly expressed in lobular carcinomas of the breast. In this report, we evaluated the biological activity of FGFR1 in a wide range of in vitro assays. Conditional activation of FGFR1 in the nontransformed MCF10A human mammary cell line, MCF10A, resulted in cellular transformation marked by epidermal growth factor-independent cell growth, anchorage-independent cell proliferation and survival, loss of cell polarity, and epithelial-to-mesenchymal transition. Interestingly, small-molecule or small interfering RNA inhibition of ribosomal S6 kinase (RSK) activity induced death of the FGFR1-transformed cells, but not of the parental MCF10A cell line. The dependence of FGFR1-transformed cells on RSK activity was further confirmed in cell lines derived from mouse and human lobular carcinomas that possess high FGFR1 activity. Taken together, these results show the transforming activity of FGFR1 in mammary epithelial cells and identify RSK as a critical component of FGFR1 signaling in lobular carcinomas, thus implicating RSK as a candidate therapeutic target in FGFR1-expressing tumors.

Lactogenic differentiation of HC11 cells is not accompanied by downregulation of AP-2 transcription factor genes.

BACKGROUND: During pregnancy the mammary epithelium undergoes a complex developmental process which culminates in the generation of the milk-secreting epithelium. Secretory epithelial cells display lactogenic differentiation which is characterized by the expression of milk protein genes, such as beta-casein or whey acidic protein (WAP). Transcription factors AP-2alpha and AP-2gamma are downregulated during lactation, and their overexpression in transgenic mice impaired the secretory differentiation of the mammary epithelium, resulting in lactation failure. To explore whether the downregulation of AP-2alpha and AP-2gamma is of functional significance for lactogenic differentiation, we analyzed the expression of the AP-2 family members during the lactogenic differentiation of HC11 mammary epithelial cells in vitro. Differentiation of HC11 cells was induced following established protocols by applying the lactogenic hormones prolactin, dexamethasone and insulin. FINDINGS: HC11 cells express all AP-2 family members except AP-2delta. Using RT-PCR we could not detect a downregulation of any of these genes during the lactogenic differentiation of HC11 cells in vitro. This finding was confirmed for AP-2alpha and AP-2gamma using Northern analysis. Differentiating HC11 cells displayed lower expression levels of milk protein genes than mammary glands of mid-pregnant or lactating mice. CONCLUSION: The extent of lactogenic differentiation of HC11 cells in vitro is limited compared to mammary epithelium undergoing secretory differentiation in vivo. Downregulation of AP-2 transcription factor genes is not required for lactogenic differentiation of HC11 cells but may functionally be involved in aspects of lactogenic differentiation in vivo that are not reflected by the HC11 system.