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Background: Immunoglobulin G and A, transferrin, haptoglobin and alpha-1- antitrypsin represent approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to the discovery of important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-glycoproteome has been traditionally utilized for diagnostic applications. Objective: Creating a five-protein model to deconstruct the overall N-glycosylation fingerprints in inflammatory and malignant lung diseases. Methods: The N-glycan pool of human serum and the five high abundant serum glycoproteins were analyzed. Simultaneous endoglycosidase/sialidase digestion was followed by fluorophore labeling and separation by CE-LIF to establish the model. Pooled serum samples from patients with COPD, lung cancer (LC) and their comorbidity were all analyzed. Results: Nine significant (>1%) asialo-N-glycan structures were identified both in human serum and the standard protein mixture. The core-fucosylated-agalacto-biantennary glycan differentiated COPD and LC and both from the control and the comorbidity groups. Decrease in the core-fucosylated-agalacto-biantennary-bisecting, monogalacto and bigalacto structures differentiated all disease groups from the control. The significant increase of the fucosylated-galactosylated-triantennary structure was highly specific for LC, to a medium extent for COPD and a lesser extent for comorbidity. Also, some increase in the afucosylated-galactosylated-biantennary structure in all three disease types and afucosylated-galactosylated-triantennary structures in COPD and LC were observed in comparison to the control group. Conclusion: Our results suggested that changes in the desialylated human serum Nglycome hold glycoprotein specific molecular diagnostic potential for malignant and inflammatory lung diseases, which can be modeled with the five-protein mixture.

Background: First-line durvalumab plus etoposide with either cisplatin or carboplatin (platinum–etoposide) showed a significant improvement in overall survival versus platinum–etoposide alone in patients with extensive-stage small-cell lung cancer (ES-SCLC) in the CASPIAN study. Here we report updated results, including the primary analysis for overall survival with durvalumab plus tremelimumab plus platinum–etoposide versus platinum–etoposide alone. Methods: CASPIAN is an ongoing, open-label, sponsor-blind, randomised, controlled phase 3 trial at 209 cancer treatment centres in 23 countries worldwide. Eligible patients were aged 18 years or older (20 years in Japan) and had treatment-naive, histologically or cytologically documented ES-SCLC, with a WHO performance status of 0 or 1. Patients were randomly assigned (1:1:1) in blocks of six, stratified by planned platinum, using an interactive voice-response or web-response system to receive intravenous durvalumab plus tremelimumab plus platinum–etoposide, durvalumab plus platinum–etoposide, or platinum–etoposide alone. In all groups, patients received etoposide 80–100 mg/m2 on days 1–3 of each cycle with investigator's choice of either carboplatin area under the curve 5–6 mg/mL/min or cisplatin 75–80 mg/m2 on day 1 of each cycle. Patients in the platinum–etoposide group received up to six cycles of platinum–etoposide every 3 weeks and optional prophylactic cranial irradiation (investigator's discretion). Patients in the immunotherapy groups received four cycles of platinum–etoposide plus durvalumab 1500 mg with or without tremelimumab 75 mg every 3 weeks followed by maintenance durvalumab 1500 mg every 4 weeks. The two primary endpoints were overall survival for durvalumab plus platinum–etoposide versus platinum–etoposide and for durvalumab plus tremelimumab plus platinum–etoposide versus platinum–etoposide in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered at ClinicalTrials.gov, NCT03043872. Findings: Between March 27, 2017, and May 29, 2018, 972 patients were screened and 805 were randomly assigned (268 to durvalumab plus tremelimumab plus platinum–etoposide, 268 to durvalumab plus platinum–etoposide, and 269 to platinum–etoposide). As of Jan 27, 2020, the median follow-up was 25·1 months (IQR 22·3–27·9). Durvalumab plus tremelimumab plus platinum–etoposide was not associated with a significant improvement in overall survival versus platinum–etoposide (hazard ratio [HR] 0·82 [95% CI 0·68–1·00]; p=0·045); median overall survival was 10·4 months (95% CI 9·6–12·0) versus 10·5 months (9·3–11·2). Durvalumab plus platinum–etoposide showed sustained improvement in overall survival versus platinum–etoposide (HR 0·75 [95% CI 0·62–0·91]; nominal p=0·0032); median overall survival was 12·9 months (95% CI 11·3–14·7) versus 10·5 months (9·3–11·2). The most common any-cause grade 3 or worse adverse events were neutropenia (85 [32%] of 266 patients in the durvalumab plus tremelimumab plus platinum–etoposide group, 64 [24%] of 265 patients in the durvalumab plus platinum–etoposide group, and 88 [33%] of 266 patients in the platinum–etoposide group) and anaemia (34 [13%], 24 [9%], and 48 [18%]). Any-cause serious adverse events were reported in 121 (45%) patients in the durvalumab plus tremelimumab plus platinum–etoposide group, 85 (32%) in the durvalumab plus platinum–etoposide group, and 97 (36%) in the platinum–etoposide group. Treatment-related deaths occurred in 12 (5%) patients in the durvalumab plus tremelimumab plus platinum–etoposide group (death, febrile neutropenia, and pulmonary embolism [n=2 each]; enterocolitis, general physical health deterioration and multiple organ dysfunction syndrome, pneumonia, pneumonitis and hepatitis, respiratory failure, and sudden death [n=1 each]), six (2%) patients in the durvalumab plus platinum–etoposide group (cardiac arrest, dehydration, hepatotoxicity, interstitial lung disease, pancytopenia, and sepsis [n=1 each]), and two (1%) in the platinum–etoposide group (pancytopenia and thrombocytopenia [n=1 each]). Interpretation: First-line durvalumab plus platinum–etoposide showed sustained overall survival improvement versus platinum–etoposide but the addition of tremelimumab to durvalumab plus platinum–etoposide did not significantly improve outcomes versus platinum–etoposide. These results support the use of durvalumab plus platinum–etoposide as a new standard of care for the first-line treatment of ES-SCLC. Funding: AstraZeneca.

Lung adenocarcinoma is one of the leading causes of mortality among cancer patients worldwide and Chronic Obstructive Pulmonary Disease (COPD) is also high in death statistics. In addition, patients with Chronic Obstructive Pulmonary Disease (COPD) have a high risk of developing primary lung cancer. Prevention, risk estimation and a non-invasive diagnostics are essential to decrease COPD and lung cancer mortality. Therefore, better and more accurate molecular diagnostic markers (biomarkers) are needed for the early differential diagnosis of these lung diseases to help clinicians make better therapeutic decisions. This review focuses on recently discovered adenocarcinoma and COPD biomarkers at the proteome and glycome level. In the first part, the protein markers are summarized, while the second part is focused on glycan markers. Their use to differentiate between chronic inflammation (COPD) and malignant (adenocarcinoma) diseases is discussed in detail.

Introduction. Numerous clinical and laboratory scores that include C-reactive protein (CRP), D-dimer, ferritin, lactate dehydrogenase (LDH), interleukin 6 (IL-6), procalcitonin (PCT), blood urea nitrogen (BUN), creatinine levels and oxygenation (PaO2 and SaO2) have been used for the prognosis of COVID-19. In addition, composite scores have been developed for the assessment of general state and risk in community-acquired pneumonia (CAP) that may be applied for COVID-19 as well. In this study, we assessed severity and potential prognostic risk factors for unfavourable outcome among hospitalized COVID-19 patients. We also applied the A-DROP general scoring system used in CAP to COVID-19. Patients and methods. Altogether 233 patients admitted to our center with COVID-19 were included in the study. Clinical status, several laboratory biomarkers described above, indicators of oxygenation were determined at hospital admission. We also applied the A-DROP composite scoring system that includes Age (≥ 70 years in males and ≥ 75 years in females), Dehydration (BUN ≥ 7.5 mmol/l), Respiratory failure (SaO2 ≤ 90% or PaO2 ≤ 60 mmHg), Orientation disturbance (confusion) and low blood Pressure (systolic BP ≤ 90 mmHg) to COVID-19. Results. At the time of admission, most patients had elevated CRP, LDH, ferritin, D-dimer and IL-6 levels indicating multisystemic inflammatory syndrome (MIS). Altogether 49 patients (21.2%) required admission to ICU, 46 (19.7%) needed ventilation and 40 patients (17.2%) died. In the binary analysis, admission to ICU, the need for ventilation and death were all significantly associated with the duration of hospitalization, history of hypertension or obesity, confusion/dizziness, as well as higher absolute leukocyte and neutrophil and lower lymphocyte counts, elevated CRP, PCT, LDH, ferritin, IL-6, BUN and creatinine levels, low PaO2 and SaO2 and higher A-DROP score at the time of admission (pConclusions. Numerous laboratory biomarkers in addition to obesity, dizziness at the time of admission and the history of hypertension may predict the need for ICU admission and ventilation, as well as mortality in COVID-19. Moreover, A-DROP may be a suitable scoring system for the assessment of general health and disease outcome in COVID-19.

Background: A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications finetune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease. Objective: The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. Methods: A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension-based method in combination with capillary gel electrophoresis was used for verification. Results: The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy–Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3’ UTR of the MGAT5 gene significantly differed between the sample groups compared. Conclusion: Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increasing branching that is the characteristic of invasive malignancies.

Absztrakt: A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnosztika révén lehetne tovább csökkenteni a COPD miatti halálozást. A fejlődő diagnosztikus technikák ellenére az optimális szűrővizsgálat felfedezése még várat magára. Kellően szenzitív és specifikus biomarkerek felfedezése megfelelő eszközt adhat a klinikus kezébe a betegség korai diagnosztizálásához, a differenciáldiagnosztikához, a fenotipizáláshoz és a prognózis becsléséhez. Közleményünkben a COPD vonatkozásában a közelmúltban felfedezett potenciális fehérje és glikán biomarkereket foglaljuk össze. Orv Hetil. 2020; 161(4): 123–128.

Absztrakt: Bevezetés: A krónikus obstruktív tüdőbetegség jelentős egészségügyi teher mind a beteg, mind a társadalom részére. Betegoktatással ez a teher csökkenthető, így magyar környezetben kerestük az optimális tartalmat ehhez. Célkitűzés: 1) A tüdőgyógyászok véleményére épülő betegoktatási tartalom tervezése; 2) a tüdőgyógyászok magatartásának és percepcióinak megismerése; 3) a beteg adherenciája (együttműködése) javítási lehetőségeinek felmérése. Módszer: Országszerte 20 gondozóban, kórházban és rehabilitációs központban dolgozó tüdőgyógyásszal készítettünk interjút. A struktúrát úgy alakítottuk ki, hogy egy betegoktató program kulcselemeit definiáljuk, valamint hogy teret engedjünk a percepciók, terápiás attitűdök felfedésének. Eredmények: Az átlagos krónikus obstruktív tüdőbeteg dohányos, férfi, alulszocializált, köhög, fullad, és 40 évnél idősebb. Betegségét nem veszi komolyan, csak súlyosabb esetben fordul orvoshoz, adherenciája is csak ekkor nő meg. Ez a hatás gyakran átmeneti, csak a rosszullétre korlátozódik. Az adherencia szerint három csoport különíthető el: marginális jó adherencia (kb. 10%) mellett az átlag 30–40% között van, és a betegek jelentős része (60%) minimális adherenciával bír. Az inhalátor használata ideális esetben maximum 3 lépésből áll, könnyen reprodukálható és elmagyarázható. Következtetés: Az oktatóprogram keretét meghatározó szempontok a megfelelő betegkép (személyre szabás), a tüdőgyógyászati, helyszíni oktatás, az orvos–beteg kapcsolat, a betegek hozzáállása és az életmódbeli változtatások (leszokás a dohányzásról), valamint a megfelelő inhalátor kiválasztása. Orv Hetil. 2020; 161(3): 95–102.

Background: Most patients with small-cell lung cancer (SCLC) have extensive-stage disease at presentation, and prognosis remains poor. Recently, immunotherapy has demonstrated clinical activity in extensive-stage SCLC (ES-SCLC). The CASPIAN trial assessed durvalumab, with or without tremelimumab, in combination with etoposide plus either cisplatin or carboplatin (platinum–etoposide) in treatment-naive patients with ES-SCLC. Methods: This randomised, open-label, phase 3 trial was done at 209 sites across 23 countries. Eligible patients were adults with untreated ES-SCLC, with WHO performance status 0 or 1 and measurable disease as per Response Evaluation Criteria in Solid Tumors, version 1.1. Patients were randomly assigned (in a 1:1:1 ratio) to durvalumab plus platinum–etoposide; durvalumab plus tremelimumab plus platinum–etoposide; or platinum–etoposide alone. All drugs were administered intravenously. Platinum–etoposide consisted of etoposide 80–100 mg/m2 on days 1–3 of each cycle with investigator's choice of either carboplatin area under the curve 5–6 mg/mL per min or cisplatin 75–80 mg/m2 (administered on day 1 of each cycle). Patients received up to four cycles of platinum–etoposide plus durvalumab 1500 mg with or without tremelimumab 75 mg every 3 weeks followed by maintenance durvalumab 1500 mg every 4 weeks in the immunotherapy groups and up to six cycles of platinum–etoposide every 3 weeks plus prophylactic cranial irradiation (investigator's discretion) in the platinum–etoposide group. The primary endpoint was overall survival in the intention-to-treat population. We report results for the durvalumab plus platinum–etoposide group versus the platinum–etoposide group from a planned interim analysis. Safety was assessed in all patients who received at least one dose of their assigned study treatment. This study is registered at ClinicalTrials.gov, NCT03043872, and is ongoing. Findings: Patients were enrolled between March 27, 2017, and May 29, 2018. 268 patients were allocated to the durvalumab plus platinum–etoposide group and 269 to the platinum–etoposide group. Durvalumab plus platinum–etoposide was associated with a significant improvement in overall survival, with a hazard ratio of 0·73 (95% CI 0·59–0·91; p=0·0047]); median overall survival was 13·0 months (95% CI 11·5–14·8) in the durvalumab plus platinum–etoposide group versus 10·3 months (9·3–11·2) in the platinum–etoposide group, with 34% (26·9–41·0) versus 25% (18·4–31·6) of patients alive at 18 months. Any-cause adverse events of grade 3 or 4 occurred in 163 (62%) of 265 treated patients in the durvalumab plus platinum–etoposide group and 166 (62%) of 266 in the platinum–etoposide group; adverse events leading to death occurred in 13 (5%) and 15 (6%) patients. Interpretation: First-line durvalumab plus platinum–etoposide significantly improved overall survival in patients with ES-SCLC versus a clinically relevant control group. Safety findings were consistent with the known safety profiles of all drugs received. Funding: AstraZeneca.

Background: Molecular response based on ctDNA dynamics may be predictive of benefit from immunotherapy in NSCLC, complementing radiologic disease assessment and potentially enabling early clinical decision-making, but data in SCLC are lacking. The phase 2, multi-arm, signal-searching BALTIC study (NCT02937818) assessed novel treatment combinations in platinum-refractory/resistant ES-SCLC; we report efficacy, safety, and exploratory ctDNA and PD-L1 analyses from pts who received D + T in Arm A. Methods: Eligible pts had ES-SCLC, progressive disease (PD) during or within 90 days of completing 1L platinum-based CT, WHO PS 0-1, and life expectancy ≥8 weeks. In Arm A, pts received D 1500 mg + T 75 mg q4w for 4 cycles, followed by maintenance D 1500 mg q4w until PD. The primary endpoint was ORR (investigator assessment, RECIST v1.1). Secondary endpoints included DCR, PFS, OS, and safety and tolerability. Prespecified exploratory analyses included assessment of baseline/on-treatment ctDNA levels measured by maximum variant allele frequency (MaxVAF), and PD-L1 expression on tumor cells (TC) and immune cells (IC) (VENTANA PD-L1 [SP263] Assay). Results: 41 pts received treatment with D + T (median age 63 years; 76% male; 76% PS 1). At the data cut-off (DCO; Jun 22, 2020), median treatment duration was 12 weeks, with 3 pts ongoing D treatment (these pts had received ≥38, ≥21, and ≥19 doses of D, respectively, at DCO). Confirmed ORR was 7.3% (95% CI 1.54‒19.92; 3 partial responses [PR]). DCR at 12 weeks was 27%. 21 pts (51%) were PD-L1 evaluable. Of 12 pts with PD-L1 expression (TC or IC) ≥1%, 3 (25%) had a best response of PR or stable disease, compared with 1 (11%) of 9 pts with PD-L1 (TC or IC) Conclusions: Although limited antitumor activity was observed in refractory/resistant ES-SCLC in BALTIC, association of lower baseline ctDNA levels (MaxVAF) with longer OS may reflect the prognostic effect of disease burden. Association of on-treatment reduction in ctDNA level with longer OS suggests potential use of ctDNA as a surrogate of treatment response in ES-SCLC. Further analyses in larger datasets and in a randomized setting are warranted. Citation Format: Niels Reinmuth, Oscar Juan-Vidal, Zsolt Horváth, Dariusz Kowalski, Anna Kryzhanivska, Eszter Csánky, Gabriella Gálffy, Maciej Bryl, David Vicente, Ihor Vynnychenko, Zsolt Pápai-Székely, Jon Armstrong, Tapashi Dalvi, Yashaswi Shrestha, Mingchao Xie, Haiyi Jiang, Igor Bondarenko. Durvalumab (D) plus tremelimumab (T) in platinum-refractory/resistant extensive-stage small cell lung cancer (ES-SCLC): Efficacy, safety and ctDNA dynamics from Arm A of the phase 2 BALTIC study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT533.

The human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 N-glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 N-linked carbohydrate structures. The classification tree analysis resulted in a panel of N-glycans, which could be used to follow up on the effects of lung tumor surgical resection.

Simple Summary Globally, there were around 2.1 million lung cancer cases and 1.8 million deaths in 2018. Hungary—where this study was carried out—had the highest rate of lung cancer in the same year. We developed a new analytical method which can be readily used to follow up the tumor surgery by investigating the glycan (sugar) structures of proteins. As the results of such investigations are very complex, computer-assisted machine learning methods were utilized for data interpretation. Abstract The human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 N-glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 N-linked carbohydrate structures. The classification tree analysis resulted in a panel of N-glycans, which could be used to follow up on the effects of lung tumor surgical resection.

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Omalizumab, a glycoprotein based biotherapeutics, is one of the most frequently used targeted antibody biopharmaceutical to reduce asthma exacerbations, improve lung function and reduce oral corticosteroid use. The effector function and clearance time of such glycoprotein drugs is affected by their N-glycosylation, that defines the required administration frequency to improve the quality of life in appropriately selected patients. Therefore, the glycosylation of biologics is an important critical quality attribute (CQA). The profile of asparagine linked carbohydrates is greatly dependent on the manufacturing process. Even a small deviation may have a major effect on the structure and therefore the function of the biotherapeutic product. For this reason, comprehensive N-glycosylation analysis is of high importance during production and release. Capillary electrophoresis (CE) is one of the frequently used tools to characterize protein therapeutics and utilized by the biopharmaceutical industry for protein and glycan level analysis, which are key parts both for drug development and quality control. To reveal important structure – function relationships, characterization of omalizumab is presented using capillary SDS gel electrophoresis with UV detection at the protein level and capillary gel electrophoresis with laser induced fluorescent detection at the N-linked carbohydrate level. This latter technique was also used for oligosaccharide sequencing for glycan structure validation. The results suggested no ADCC function – structure relationship due to the mostly core fucosylated biantennary glycans found. However, the presence of the high mannose structures probably affects the clearance rate of the drug.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.

Chronic obstructive pulmonary disease (COPD) is worldwide a significant representative of morbidity and mortality statistics. COPD is a preventable and treatable disease and smoking is the main risk factor of disease development. Prevention is crucial, but it has its limitations, so risk estimation and early non-invasive diagnostics are essential to decrease COPD mortality. Although diagnostic techniques are evolving, the perfect screening tool is lacking. Discovery of properly sensitive and specific biomarkers is important. They could be effective diagnostic, differential diagnostic, phenotyping and prognostic tools to clinicians. The manuscript is focusing on recently discovered potential protein and glycan biomarkers for COPD. Orv Hetil. 2020; 161(4): 123-128.Absztrakt: A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnosztika révén lehetne tovább csökkenteni a COPD miatti halálozást. A fejlődő diagnosztikus technikák ellenére az optimális szűrővizsgálat felfedezése még várat magára. Kellően szenzitív és specifikus biomarkerek felfedezése megfelelő eszközt adhat a klinikus kezébe a betegség korai diagnosztizálásához, a differenciáldiagnosztikához, a fenotipizáláshoz és a prognózis becsléséhez. Közleményünkben a COPD vonatkozásában a közelmúltban felfedezett potenciális fehérje és glikán biomarkereket foglaljuk össze. Orv Hetil. 2020; 161(4): 123–128.

Absztrakt

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important finding was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.

Multiple myeloma (MM) is an immedicable malignancy of the human plasma cells producing abnormal antibodies (also referred to as paraproteins) leading to kidney problems and hyperviscosity syndrome. In this paper, we report on the N-glycosylation analysis of paraproteins from total human serum as well as the fragment crystallizable region (Fc ) and fragment antigen binding (Fab ) κ/λ light chain fractions of papain digested immunoglobulins from multiple myeloma patients. CE-LIF detection was used for the analysis of the N-glycans after endoglycosidase (PNGase F) mediated sugar release and fluorophore labeling (APTS). While characteristic N-glycosylation pattern differences were found between normal control and untreated, treated and remission stage multiple myeloma patient samples at the global serum level, less distinctive changes were observed at the immunoglobulin level. Principal component analysis adequately differentiated the four groups (control and three patient groups) on the basis of total serum N-glycosylation analysis. 12 N-glycan features showed statistically significant differences (p

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.

A tüdőtranszplantáció széles körben elfogadott kezelési lehetőség végstádiumú tüdőbetegek számára, akiknek a funkcionális státusza maximális kezelés mellett romlik. A szerzők egy fiatal betegük esetét ismertetik, aki végstádiumú idiopathiás tüdőfibrosis miatt Bécsben tüdőtranszplantáción esett át. Az elhúzódó, szövődményes posztoperatív szakban akut veseelégtelenség alakult ki, emiatt a beteg hemodialízisre szorult. Pulmonológiai szempontból teljes rehabilitációja valósult meg, és a rendszeres művesekezelés az opportunista fertőzések kialakulásának a kockázatát jelentős mértékben megnövelte, a beteg életminőségét is jelentősen rontotta, ezért négy évvel a tüdőtranszplantáció után vesetranszplantációra került sor Debrecenben. Ez az első magyar tüdőtranszplantált beteg, aki Magyarországon veseátültetésen is átesett. Több mint két évvel a vesetranszplantáció után a beteg kompenzáltan, rehabilitáltan él, légzésfunkciós értékei tovább javultak, és vesefunkciós paraméterei is kielégítőek. Az elmúlt évek során szignifikánsan megnövekedett a különböző szervtranszplantáción áteső betegek száma világszerte és hazánkban is. Ezeknél a betegeknél elsősorban az immunszuppresszív szerek, calcineurininhibitorok hatására számtalan szövődménnyel kell számolni. Kiemelendő a szekunder veseelégtelenség, amely mind akut, mind krónikus formában jelentkezhet, és akár vesetranszplantáció indikációját is képezheti. A veseátültetés kiváló kezelési lehetőség olyan végstádiumú vesebetegek számára, akik korábban más szervtranszplantáción estek át. A veseátültetés javítja az életminőséget, és a várható túlélést is növeli.

We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua . Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua .

The authors present the case of a 72-year-old woman who underwent coronary bypass grafting. Left sided chylothorax due to accidental dissection of a thoracic duct branch developed 2 months after sternotomy. As conservative therapy has failed, surgical pleurodesis was performed successfully. Chylothorax is a rare and underestimated complication of coronary bypass grafting. The worldwide increasing number of coronary artery bypass grafting surgeries makes it important to pay attention to this condition. Thus diagnosis of the chyle is relatively easy by its high chylomicron and triglyceride content, but identification of the etiology and its treatment is sometimes challenging for the physician. The treatment of chylothorax is usually conservative. The main goal is to keep the volume of the chyle under control. The number of surgical interventions because of chylothorax is increasing due to an increase of iatrogenic etiology. Orv. Hetil., 2012, 153, 553–558.

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.

Background: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression. Objectives: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment. Method: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction. Results: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV1. Conclusion: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression.

Background In addition to smoking, genetic predisposition is believed to play a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Genetic association studies of new candidate genes in COPD may lead to improved understanding of the pathogenesis of the disease. Methods Two proposed casual single nucleotide polymorphisms (SNP) (rs1051740, rs2234922) in microsomal epoxide hydrolase (EPHX1) and three SNPs (rs1801282, rs1800571, rs3856806) in peroxisome proliferator-activated receptor gamma (PPARG), a new candidate gene, were genotyped in a case-control study (272 COPD patients and 301 controls subjects) in Hungary. Allele frequencies and genotype distributions were compared between the two cohorts and trend test was also used to evaluate association between SNPs and COPD. To estimate the strength of association, odds ratios (OR) (with 95% CI) were calculated and potential confounding variables were tested in logistic regression analysis. Association between haplotypes and COPD outcome was also assessed. Results The distribution of imputed EPHX1 phenotypes was significantly different between the COPD and the control group (P = 0.041), OR for the slow activity phenotype was 1.639 (95% CI = 1.08- 2.49; P = 0.021) in our study. In logistic regression analysis adjusted for both variants, also age and pack-year, the rare allele of His447His of PPARG showed significant association with COPD outcome (OR = 1.853, 95% CI = 1.09-3.14, P = 0.0218). In haplotype analysis the GC haplotype of PPARG (OR = 0.512, 95% CI = 0.27-0.96, P = 0.035) conferred reduced risk for COPD. Conclusions The "slow" activity-associated genotypes of EPHX1 were associated with increased risk of COPD. The minor His447His allele of PPARG significantly increased; and the haplotype containing the minor Pro12Ala and the major His447His polymorphisms of PPARG decreased the risk of COPD.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

The effect of alkaline pH and alkaline hydrolysis on the physico-chemical and biological properties of endotoxin (ET) isolated from Serratia marcescens ATCC 13477 by the Biovin procedure was studied. Major emphasis was put on the ion exchange column chromatography and immune adjuvant activity (ADA) of the alkali treated samples. To measure changes in some endotoxicity parameters, Limulus lysate clotting (LAL), chick embryo lethality, Shwartzman skin reactivity and in vitro TNF release were measured. The toxic properties of ET, with the unique exception of the Shwartzman skin reactivity, rapidly diminished during alkaline treatment. As immunogen CBre3, a recombinant HIV glycoptotein which spans the C terminus of gp 120 and the N terminus of gp 41, was used in CD-1 mice, alkali treated and immediately neutralized ET samples (zero time) were inactive as adjuvants, in some cases immunosuppression could be clearly seen. But if the alkaline hydrolysis was continued for 6 h, the ADA became higher than it had been for the starting ET sample. Further alkaline hydrolysis eliminated the ADA of the samples. Both NaOH and propylamine acted similarly on the ET preparation. Reaction kinetic studies of the NaOH detoxification indicated the cleavage of ester bound acyl groups with low binding energy. Chemical analyses of the samples revealed that changes occured in the fatty acid composition, characterized by a loss of approximately half of the 3-OH myristic acid content.

Expression of microRNAs (miRNAs) is characteristically altered in cancer, and they may play a role in cancer development and progression. The authors performed microarray and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses to determine the miRNA expression profile of primary small cell lung cancer. Here we show that at least 24 miRNAs are differentially expressed between normal lung and primary small cell lung cancer (SCLC) tumors. These include miR-301, miR-183/96/182, miR-126, and miR-223, which are microRNAs deregulated in other tumor types as well; and other miRNAs, such as miR-374 and miR-210, not previously reported in association with lung cancer. The aberrant miRNA profile of SCLC may offer new insights in the biology of this aggressive tumor, and could potentially provide novel diagnostic markers.

The authors present a case report of a 17 years old female patient who had a peripheral round shadow on the chest X-ray. After the operation the histological examination verified cystic adenomatoid malformation. The cystic adenomatoid malformation is an abnormal growth of the terminal bronchiolar structures. It is a rare disease, but often associates with other developmental abnormalities. It is important in the differential diagnosis to separate this disease from other cystic pulmonary lesions or sequestration. It is usually recognized in antenatal period or early childhood, but rarely in adulthood. It is possible that there is a connection between the adenoid malformation and lung cancer.

The role of volume reduction surgery in the therapy of early stage chronic obstructive pulmonary disease. COPD is a leading cause of morbidity and mortality worldwide. Assessment of COPD severity is based on the level of patient's symptoms, the severity of the spirometric abnormality and the presence of complications, such as respiratory failure, and right heart failure. Management of mild to moderate COPD is complex, involves the avoidance of risk factors to prevent disease progression, pharmacotherapy as needed to control symptoms, physiotherapy, and rehabilitation. The bases of pharmacotherapy are bronchodilators, which represent the first line symptom based pharmacologic intervention in COPD. Surgical treatment of emphysema was basically designed to improve subjective dyspnoea, respiratory function and quality of life in severely disabled patients. Three surgical options are currently employed, namely bullectomy, lung volume surgery and lung transplantation. During volume reduction surgery the hyperinflated areas are resected, so the functional residual volume is getting reduced, the respiratory muscle function is increased; the quality of life is improved. The authors are report a young early stage COPD patient who underwent a volume reduction surgery because of decline in physical condition, improving her disability. In conclusion in properly selected patients, lung volume surgery can offer prolonged functional benefit and satisfactory long term survival, even in early stage. The invasive options has a risk of both mortality and morbidity, they are directed only at patients who remain symptomatic despite optimal medical treatment.

Lung transplantation is now generally accepted as a useful modality of care for patients with severe life-threatening respiratory diseases that are refractory to conventional therapies. Lung transplantation is indicated for patients with end stage lung diseases, who demonstrate declining function despite maximal therapy. The author reports own experience of selection, timing referrals for lung transplantation, and the management of patients on the waiting list. Eleven patients were selected for lung transplantation. Eight of them suffered from end stage pulmonary fibrosis, two of them cystic fibrosis, and one patient had pulmonary hypertension. Seven patients underwent a successful lung transplantation and four patients died during the investigations, before the operation, due to respiratory failure, and pulmonary heart disease. It is generally recommended to consider transplantation when patients who suffer in the end stage pulmonary disease are symptomatic during daily activities too, due to pulmonary status and the expected survival is to be strongly limited, less than two years.

The pathogenesis of infiltrative pulmonary diseases is largely unknown, and their final outcome is variable. These inflammatory processes may involve the alveoli and the pulmonary interstices, and they may lead to pulmonary fibrosis. The development of end-stage pulmonary disease is irreversible. Patients with respiratory failure have a poor prognosis. For young patients at the end-stage of their pulmonary disease, lung transplantation is a promising therapeutic modality. The therapeutic protocols for unilateral or bilateral lung transplantation and heart-lung transplantation were established only in the last 10 years. In Hungary there is no center yet performing lung transplantation. This therapy is available only for those who could be effectively rehabilitated with this method. Four patients 27-35 years of age suffering pulmonary fibrosis, despite their young age developed honey-comb lungs with therapy resistant respiratory failure. No other organic symptoms were detected by detailed examination, so their predicted survival was dependent on their pulmonary status alone. Cases of four young patients, who were effectively treated with lung transplantation, are discussed from pre-operative preparations to post-operative follow-up, with the review of the literature.