Label-Free Glycopeptide Quantification for Biomarker Discovery in Human Sera

Glycan moieties of glycoproteins modulate many biological processes in mammals, such as immune response, inflammation, and cell signaling. Numerous studies show that many human diseases are correlated with quantitative alteration of protein glycosylation. In some cases, these changes can occur for certain types of glycans over specific sites in a glycoprotein rather than on the global abundance of the glycoprotein. Conventional analytical techniques that analyze the abundance of glycans cleaved from glycoproteins cannot reveal these subtle effects. Here we present a novel statistical method to quantify the site-specific glycosylation of glycoproteins in complex samples using label-free mass spectrometric techniques. Abundance variations between sites of a glycoprotein as well as different glycoforms, that is, glycopeptides with different glycans attached to the same site, can be detected using these techniques. We applied our method to an esophageal cancer study based on blood serum samples from cancer patients in an attempt to detect potential biomarkers of site-specific N-linked glycosylation. A few glycoproteins, including vitronectin, showed significantly different site-specific glycosylations within cancer/control samples, indicating that our method is ready to be used for the discovery of glycosylated biomarkers

Sequence variants and mutations in genes associated with MDS and FAO.

<p>Sequence variants and mutations in genes associated with MDS and FAO.</p

Clinical characteristics of subjects with indeterminate ALF stratified by blood molar L/P ratio at presentation.

<p>Clinical characteristics of subjects with indeterminate ALF stratified by blood molar L/P ratio at presentation.</p

Liver histology, ultrastructure, mtDNA copy number and respiratory complex activity of subjects with heterozygous mutations in <i>RRM2B</i>.

<p><b>(A)</b> Liver biopsy following the second episode of ALF in subject ID#11 showed mild steatosis. H&E stain. <b>(B)</b> Mitochondria at that time were normal in size with pale matrix and subtle haphazard arrangement of cristae. <b>(C</b> and <b>D)</b> Random, non-zonal macro- and microvesicular steatosis was prevalent in H&E- stained sections from the explanted livers of both subjects, confirmed with Oil red O in a frozen section (<b>C, insert).</b> Both explanted livers contained unusual necrotic/apoptotic hepatocytes with distorted contours, granular eosinophilic cytoplasm, and fat droplets. (<b>E</b>) Copy numbers of mtDNA in liver tissues from age-matched control subjects without ALF and from subjects #3 and #11 were determined by qPCR for the mitochondrial genes <i>CytB</i> and <i>Cox2</i>, normalized to the nuclear gene <i>B2M</i>. (<b>F</b>) Respiratory chain complex activities were simultaneously determined on frozen samples from explanted livers of ID#3 and ID#11 and from liver tissue of control subjects without ALF. (<b>G</b>)Total protein extracts from liver tissues from ID#11and from two age-matched controls without ALF were analyzed by SDS-PAGE and immunoblotting against p53R2, the 39kDa gene product of <i>RRM2B</i>.</p

Isolated complex 1 deficiency in an infant with compound heterozygous mutations in <i>ACAD9</i>.

<p>Subject #12 harboring the variant L314P and the mutation E63X in <i>ACAD9</i> succumbed to ALF and multi-organ failure within the first 24 hours of life. Post-mortem, liver architecture was preserved without significant collapse or inflammation and with minimal steatosis (H&E, <b>A</b>). However, abnormally granular hepatocytes (H&E, <b>B</b>) and mitochondrial hyperplasia detected by immunohistochemistry against mitochondrial antigens (<b>C</b>) suggest a mitochondrial hepatopathy. Respiratory chain complex assay demonstrates reduced activity of complex 1 in frozen liver, compared to liver from controls without ALF (<b>D</b>).</p

Summary of clinical course for the 12 subjects enrolled into targeted NGS.

<p>Summary of clinical course for the 12 subjects enrolled into targeted NGS.</p

Clinical characteristics of study subjects with acute liver failure.

<p>Clinical characteristics of study subjects with acute liver failure.</p

The genomic and clinical consequences of replacing procarbazine with dacarbazine in escalated BEACOPP for Hodgkin lymphoma: a retrospective, observational study

Background: Procarbazine-containing chemotherapy regimens are associated with cytopenias and infertility, suggesting stem-cell toxicity. When treating Hodgkin lymphoma, procarbazine in escalated-dose bleomycin–etoposide–doxorubicin–cyclophosphamide–vincristine–procarbazine–prednisolone (eBEACOPP) is increasingly replaced with dacarbazine (eBEACOPDac) to reduce toxicity. We aimed to investigate the impact of this drug substitution on the mutation burden in stem cells, patient survival, and toxicity.Methods: In this two-part retrospective, observational study, we first compared mutational landscapes in haematopoietic stem and progenitor cells (HSPCs) from patients with advanced-stage Hodgkin lymphoma in remission for at least 6 months who had been treated with eBEACOPDac (eBEACOPDac cohort), eBEACOPP (real-world eBEACOPP cohort), or doxorubicin–bleomycin–vinblastine–dacarbazine (ABVD); in buccal DNA from five children of a female patient with classical Hodgkin lymphoma treated with eBEACOPP before conceiving the third child; in sperm DNA from a patient with mild oligospermia treated with eBEACOPP; and in caecal adenocarcinoma and healthy colon tissue from a survivor of Hodgkin lymphoma treated with chlorambucil–vinblastine–procarbazine–prednisolone. For the second part, we analysed efficacy and toxicity data from adult patients (aged >16 years) treated with first-line eBEACOPDac (eBEACOPDac cohort) at 25 centres across UK, Ireland, and France; efficacy was compared with the German HD18 eBEACOPP trial data and toxicity with a UK real-world dataset. Participants in the German HD18 and UK real-world datasets were adults (aged >16 years) with previously untreated Hodgkin lymphoma, treated with first-line eBEACOPP. We had two co-primary objectives: to define the comparative stem-cell mutation burden and mutational signatures after treatment with or without procarbazine-containing chemotherapy (first study part); and to determine progression-free survival of patients with Hodgkin lymphoma treated with eBEACOPP or eBEACOPDac (second study part). Secondary objectives included overall survival and explored differences in specific toxicity outcomes, including transfusion requirements and measures of reproductive health (second study part).Findings: In the first part of the study (mutational analysis), patients treated with eBEACOPP (n=5) exhibited a higher burden of point mutations in HSPCs compared with those treated with eBEACOPDac (n=4) or ABVD (n=3; excess mutations 1150 [95% CI 934–1366] vs 290 [241–339] vs 186 [116–254]). Two novel mutational signatures, SBSA (SBS25-like) and SBSB, were identified in HSPCs and in a single neoplastic and healthy colon sample from patients who received procarbazine-containing chemotherapy. SBSB was also identified in germline DNA of three children conceived after eBEACOPP and in sperm of a male patient treated with eBEACOPP. SBSC was detected in patients treated with either ABVD or eBEACOPDac. In the second part of the study (efficacy and toxicity analysis), dacarbazine substitution did not appear to compromise efficacy or safety. 312 patients treated with eBEACOPDac (eBEACOPDac cohort; treated 2017–22, 186 [60%] male, median follow-up 36·0 months [IQR 25·2–50·1]) had a 3-year progression-free survival of 93·3% (95% CI 90·3–96·4), which was similar to the 93·3% [95% CI 92·1–94·4]) progression-free survival seen in 1945 patients in the German HD18 eBEACOPP trial (treated 2008–14, 1183 [61%] male, median follow-up 57·0 months [35·4–64·7]). Patients treated with eBEACOPDac required fewer blood transfusions (mean 1·70 units [SD 2·77] vs 3·69 units [3·89]; pInterpretation: Procarbazine induces a higher mutation burden and novel mutational signatures in patients with Hodgkin lymphoma treated with eBEACOPP and their germline DNA, raising concerns for the genomic health of survivors of Hodgkin lymphoma and hereditary consequences for their offspring. However, replacing procarbazine with dacarbazine appears to mitigate gonadal and stem-cell toxicity while maintaining similar clinical efficacy.</p