Reference Values of Ceruloplasmin across the Adult Age Range in a Large Italian Healthy Population.

BACKGROUND: Ceruloplasmin (Cp) is the most important serum copper transport protein playing a key role in the binding of iron to transferrin. It is a positive acute-phase response protein and the first-level diagnostic marker for Wilson disease and aceruloplasminemia. However, standardization of Cp measurement has not been successful, and assay specific reference levels of Cp are required. METHODS: From May 2019 to July 2022, we enrolled 1706 consecutive healthy Italian blood donors (1285 men and 421 women, 18 to 65 years) to identify the reference intervals of serum Cp through quantile regression and evaluate the relationship of Cp with age, sex, iron, and metabolic status through linear regression. RESULTS: We found that mean serum Cp was influenced by sex and slightly by age. The lower reference Cp value rose slightly with increasing age in both men and women. The upper reference value increased, reaching a plateau of about 25 mg/dL around 25 years in men, while in women it initially increased to around 45 mg/dL in young adults to fall sharply below 30 mg/dL for adults after their fifties. CONCLUSIONS: We showed that the normal reference curves of serum Cp vary according to sex in a large population of healthy adults. While the lower reference values did not appear to be influenced by age and sex, the upper ones differed according to sex and age showing a particularly high variability in women, possibly reflecting different hormonal status.

Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Identification and confirmation via in situ hybridization of Merkel cell polyomavirus in rare cases of posttransplant cutaneous T-cell lymphoma.

BACKGROUND: Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens. METHODS: Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip. RESULTS: Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes. CONCLUSIONS: Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.

Realization of thousand-second improved confinement plasma with Super I-mode in Tokamak EAST.

Mastering nuclear fusion, which is an abundant, safe, and environmentally competitive energy, is a great challenge for humanity. Tokamak represents one of the most promising paths toward controlled fusion. Obtaining a high-performance, steady-state, and long-pulse plasma regime remains a critical issue. Recently, a big breakthrough in steady-state operation was made on the Experimental Advanced Superconducting Tokamak (EAST). A steady-state plasma with a world-record pulse length of 1056 s was obtained, where the density and the divertor peak heat flux were well controlled, with no core impurity accumulation, and a new high-confinement and self-organizing regime (Super I-mode = I-mode + e-ITB) was discovered and demonstrated. These achievements contribute to the integration of fusion plasma technology and physics, which is essential to operate next-step devices.

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

Molecular Monitoring of Lymphomas.

Molecular monitoring of tumor-derived alterations has an established role in the surveillance of leukemias, and emerging nucleic acid sequencing technologies are likely to similarly transform the clinical management of lymphomas. Lymphomas are well suited for molecular surveillance due to relatively high cell-free DNA and circulating tumor DNA concentrations, high somatic mutational burden, and the existence of stereotyped variants enabling focused interrogation of recurrently altered regions. Here, we review the clinical scenarios and key technologies applicable for the molecular monitoring of lymphomas, summarizing current evidence in the literature regarding molecular subtyping and classification, evaluation of treatment response, the surveillance of active cellular therapies, and emerging clinical trial strategies.

Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA.

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

Short Diagnosis-to-Treatment Interval Is Associated With Higher Circulating Tumor DNA Levels in Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias. PATIENTS AND METHODS: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome. RESULTS: Short DTI was associated with advanced-stage disease (P < .001) and higher IPI (P < .001). We also found an inverse correlation between DTI and TMTV (RS = -0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels (P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI (P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2]). CONCLUSION: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials.

Droplet Digital PCR Improves IG-/TR-based MRD Risk Definition in Childhood B-cell Precursor Acute Lymphoblastic Leukemia.

Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real-time quantitative polymerase chain reaction (RQ-PCR) represents the gold standard for molecular MRD assessment and risk-based stratification of front-line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurth-Munschen (BFM) group AIEOP-BFM ALL2009 and ALL2017, B-lineage ALL patients with high RQ-PCR-MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP-BFM ALL2000 study, these patients are treated as high-risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ-PCR ("positive not-quantifiable," POS-NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients' risk definition, we analyzed MRD in 209 pediatric B-lineage ALL cases classified by RQ-PCR as POS-NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP-BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ-PCR MRD ≥ 5.0 × 10-4 at day+33 and POS-NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients' subgroup, while ddPCR NEG or ddPCR-POS-NQ patients had a significantly better outcome (P < 0.001). Overall, in 112 MR cases and 52 standard-risk patients, MRD negativity and POS-NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ-PCR in the measurement of MRD, particularly in late follow-up time points, and may thus allow improving patients' stratification in ALL protocols.

p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

Early loss of mitochondrial complex I and rewiring of glutathione metabolism in renal oncocytoma.

Renal oncocytomas are benign tumors characterized by a marked accumulation of mitochondria. We report a combined exome, transcriptome, and metabolome analysis of these tumors. Joint analysis of the nuclear and mitochondrial (mtDNA) genomes reveals loss-of-function mtDNA mutations occurring at high variant allele fractions, consistent with positive selection, in genes encoding complex I as the most frequent genetic events. A subset of these tumors also exhibits chromosome 1 loss and/or cyclin D1 overexpression, suggesting they follow complex I loss. Transcriptome data revealed that many pathways previously reported to be altered in renal oncocytoma were simply differentially expressed in the tumor's cell of origin, the distal nephron, compared with other nephron segments. Using a heuristic approach to account for cell-of-origin bias we uncovered strong expression alterations in the gamma-glutamyl cycle, including glutathione synthesis (increased GCLC) and glutathione degradation. Moreover, the most striking changes in metabolite profiling were elevations in oxidized and reduced glutathione as well as γ-glutamyl-cysteine and cysteinyl-glycine, dipeptide intermediates in glutathione biosynthesis, and recycling, respectively. Biosynthesis of glutathione appears adaptive as blockade of GCLC impairs viability in cells cultured with a complex I inhibitor. Our data suggest that loss-of-function mutations in complex I are a candidate driver event in renal oncocytoma that is followed by frequent loss of chromosome 1, cyclin D1 overexpression, and adaptive up-regulation of glutathione biosynthesis.

Exome Sequencing of African-American Prostate Cancer Reveals Loss-of-Function ERF Mutations.

African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (n = 102) and targeted validation (n = 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in ERF, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed ERF deletions in 3% of primary prostate cancers and mutations or deletions in ERF in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of ERF confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that ERF is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.Significance: Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of ERF as a prostate cancer gene; somatic copy-number alteration differences; and uncommon PIK3CA and PTEN alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies. Cancer Discov; 7(9); 973-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study.

PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.

The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine.

BACKGROUND: The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries. METHODS: We modeled common tumor profiling modalities-large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels-using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection. RESULTS: After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher's exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r(2) = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r(2) = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load. CONCLUSIONS: Large tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities.

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.

BACKGROUND: Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. METHODS: We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. RESULTS: A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). CONCLUSIONS: In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

S-phase-dependent p50/NF-кB1 phosphorylation in response to ATR and replication stress acts to maintain genomic stability.

The apical damage kinase, ATR, is activated by replication stress (RS) both in response to DNA damage and during normal S-phase. Loss of function studies indicates that ATR acts to stabilize replication forks, block cell cycle progression and promote replication restart. Although checkpoint failure and replication fork collapse can result in cell death, no direct cytotoxic pathway downstream of ATR has previously been described. Here, we show that ATR directly reduces survival by inducing phosphorylation of the p50 (NF-κB1, p105) subunit of NF-кB and moreover, that this response is necessary for genome maintenance independent of checkpoint activity. Cell free and in vivo studies demonstrate that RS induces phosphorylation of p50 in an ATR-dependent but DNA damage-independent manner that acts to modulate NF-кB activity without affecting p50/p65 nuclear translocation. This response, evident in human and murine cells, occurs not only in response to exogenous RS but also during the unperturbed S-phase. Functionally, the p50 response results in inhibition of anti-apoptotic gene expression that acts to sensitize cells to DNA strand breaks independent of damage repair. Ultimately, loss of this pathway causes genomic instability due to the accumulation of chromosomal breaks. Together, the data indicate that during S-phase ATR acts via p50 to ensure that cells with elevated levels of replication-associated DNA damage are eliminated.