CYFRA21-1 is a more sensitive biomarker to assess the severity of pulmonary alveolar proteinosis.

BACKGROUND: Serum lactate dehydrogenase (LDH), carcinoembryonic antigen (CEA) and CYFRA21-1 are the commonly used biomarkers to identify patients with autoimmune pulmonary alveolar proteinosis (APAP). However, it is not clear which of the biomarkers is more sensitive to the severity of the patient's condition. METHODS: APAP patients numbering 151 were enrolled in this study. All patients' severity was assessed through the severity and prognosis score of PAP (SPSP). According to the respective laboratory upper limits of serum levels of LDH, CEA and CYFRA21-1, APAP patients were divided into higher and lower-level groups. Patients were divided into five groups based on SPSP. 88 patients had completed six months of follow-up. We calculated sensitivity, specificity, and critical point of LDH, CEA and CYFRA21-1 between APAP patients and normal control group, and between grade 1-2 and 3-5 through receiving operating characteristics (ROC) curve. RESULTS: Serum LDH, CEA and CYFRA21-1 levels of patients with PAP were higher and distinctly related to PaO2, FVC, FEV1, DLCO, HRCT scores and SPSP. The SPSP of patients in higher-level LDH, CEA and CYFRA21-1 groups were higher than those of corresponding lower-level groups. Based on SPSP results, the patients were divided into five groups (grade I, 20; grade II, 37; grade III, 40; grade IV, 38; grade V, 16). The serum level of CYFRA21-1 of patients with APAP in grade II was higher than that of patients in grade I and lower than that of patients in grade III. Serum CYFRA21-1 of patients with APAP after six months were higher than the baseline among the aggravated group. Serum LDH, CEA and CYFRA21-1 levels after six months among patients in the relieved group of patients with APAP were lower than the baseline. ROC correlating LDH, CEA and CYFRA21-1 values with APAP severity (between grade 1-2 and 3-5) showed an optimal cutoff of LDH of over 203 U/L (< 246 U/L), CEA of over 2.56 ug/L (< 10 ug/L), and CYFRA21-1 of over 5.57 ng/ml (> 3.3 ng/ml) (AUC: 0.815, 95% CI [0.748-0.882], sensitivity: 0.606, specificity: 0.877). CONCLUSION: Serum CYFRA21-1 level was more sensitive in revealing the severity of APAP than LDH and CEA levels among mild to moderate forms of disease.

Autoimmune Pulmonary Alveolar Proteinosis Complicated with Sarcoidosis: the Clinical Course and Serum Levels of Anti-granulocyte-macrophage colony-stimulating Factor Autoantibody.

Autoimmune pulmonary alveolar proteinosis (APAP) is caused by macrophage dysfunction due to anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody. We experienced 2 cases of APAP complicated with sarcoidosis in a 42-year-old woman and a 51-year-old man (age at the sarcoidosis diagnosis). APAP preceded sarcoidosis in the woman, and both diseases were diagnosed simultaneously in the man. Sarcoidosis lesions were observed in the lung, skin, and eyes, and the pathological findings of APAP were not marked at the diagnosis of sarcoidosis in either case. Low-grade positive serum anti-GM-CSF autoantibody was suspected to be correlated with the occurrence of sarcoidosis and resolution of APAP.

Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis.

Recently, attempts to reveal the structures of autoantibodies comprehensively using improved proteogenomics technology, have become popular. This technology identifies peptides in highly purified antibodies by using an Orbitrap device to compare spectra from liquid chromatography-tandem mass spectrometry against a cDNA database obtained through next-generation sequencing. In this study, we first analyzed granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies in a patient with autoimmune pulmonary alveolar proteinosis, using the trapped ion mobility spectrometry coupled with quadrupole time-of-flight (TIMS-TOF) instrument. The TIMS-TOF instrument identified peptides that partially matched sequences in up to 156 out of 162 cDNA clones. Complementarity-determining region 3 (CDR3) was fully and partially detected in nine and 132 clones, respectively. Moreover, we confirmed one unique framework region 4 (FR4) and at least three unique across CDR3 to FR4 peptides via de novo peptide sequencing. This new technology may thus permit the comprehensive identification of autoantibody structure.

Valuable Serum Markers in Pulmonary Alveolar Proteinosis.

OBJECTIVE: Several serum markers were reported to reflect the severity of pulmonary alveolar proteinosis (PAP). The aim of this study is to investigate a reliable and facile marker to access and monitor the clinical course of PAP in a large cohort. METHODS: PAP patients from January 2010 to June 2018 were enrolled. Hospital records were used as data sources. The levels of various serum indicators were detected. We evaluated the correlation between pulmonary function test results and clinical variables. RESULTS: Diffusion capacity for carbon monoxide (DLCO) level was positively correlated with the level of high-density lipoprotein cholesterol (HDL-C) (P < 0.05) in 122 patients of PAP at baseline. The levels of HDL-C and DLCO significantly increased while carcinoembryonic antigen (CEA), CYFRA21-1, neuron-specific enolase (NSE), and lactic dehydrogenase (LDH) levels decreased six months after granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation therapy between 14 patients with PAP. Nevertheless, the increased DLCO was significantly correlated with decreased CEA (r = -0.579, P = 0.031) and CYFRA 21-1 (r = -0.632, P = 0.015). In 10 PAP patients without GM-CSF inhalation therapy, HDL-C and DLCO significantly decreased while NSE and LDH levels increased after six months of follow-up. The decreased DLCO was significantly correlated with increased LDH (r = -0.694, P = 0.026). CONCLUSIONS: Serum CEA, CYFRA21-1, and LDH are valuable serum markers for the evaluation of disease activity of PAP and may predict the response to treatment of PAP.

Memory B cell pool of autoimmune pulmonary alveolar proteinosis patients contains higher frequency of GM-CSF autoreactive B cells than healthy subjects.

The IgG-type neutralizing GM-CSF autoantibody (GMAb) is known to be the causative agent for autoimmune pulmonary alveolar proteinosis (APAP). Previous studies report that serum levels of IgG-GMAb are approximately 50-fold higher in APAP patients than in healthy subjects (HS). Serum levels of IgM-GMAb are also higher in APAP patients than in HS, but this has been assumed to be an etiological bystander. However, the mechanism for the excessive production of IgG-GMAb in APAP remains unclear. To investigate this, we detected putative GMAb-producing B cells (PGMPB) by inoculated B cells from the peripheral blood of APAP patients, HS, and umbilical cord blood mononuclear cells (UCBMNs) with Epstein-Barr virus. Both ELISA and ELISPOT assays showed that IgM-type GMAb was consistently and frequently present in all three groups, whereas IgG-type GMAb was high only in APAP patients, in whom it was exclusively produced in memory B cells and not in naive B cells. Since PGMPB in UCBMNs produced IgM-GMAb, but not IgG-GMAb, to the same extent as in HS and APAP patients, most IgM-GMAb reacted with GM-CSF in a non-specific manner. The memory B cell pool of APAP patients contain higher frequency of PGMPB than that of healthy subjects.

Assay system development to measure the concentration of sargramostim with high specificity in patients with autoimmune pulmonary alveolar proteinosis after single-dose inhalation.

During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), sargramostim, in patients with autoimmune pulmonary alveolar proteinosis (aPAP), we conducted a pharmacokinetic study of single-dose sargramostim inhalation. Several problems were encountered whereby sargramostim formed an immune-complex with GM-CSF autoantibodies (GMAbs) immediately after entering the body; thus, we could not measure the concentration of sargramostim using a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA). Moreover, the ELISA could not discriminate inhaled sargramostim from intrinsic GM-CSF. To solve these problems, we developed a novel ELISA system with a capture antibody that is specific for sargramostim and a detection antibody capable of binding with GM-CSF. This system quantified the serum sargramostim concentration, but not E. coli-, CHO-, or HEK293T-derived human recombinant GM-CSF. Using this system, serum pharmacokinetics were estimated in five patients after inhalation of 250 µg sargramostim, with a mean Cmax of 9.7 ±â€¯2.85 pg/ml at a Tmax of 2 ±â€¯1.22 h.

Elevated Serum Anti-GM-CSF Antibodies before the Onset of Autoimmune Pulmonary Alveolar Proteinosis in a Patient with Sarcoidosis and Systemic Sclerosis.

Pulmonary alveolar proteinosis (PAP) is characterized by the accumulation of periodic acid-schiff stain-positive lipoproteinaceous materials in the alveolar space due to impaired surfactant clearance by alveolar macrophage. Autoimmune PAP is the most common form of PAP, but rarely accompanies collagen disease or sarcoidosis. We report here a rare case of autoimmune PAP preceded by systemic sclerosis and sarcoidosis. A 64-year-old woman was admitted to our hospital for blurred vision, muscle weakness of extremities, Raynaud's phenomenon, and exertional dyspnea. We diagnosed her as having systemic sclerosis complicated with sarcoidosis. Chest computed tomography (CT) and transbronchial lung biopsy showed the findings of pulmonary fibrosis without PAP. We treated her with corticosteroid and intravenous cyclophosphamide therapy, followed by tacrolimus therapy. Thereafter, her symptoms improved except for exertional dyspnea, and she began to complain of productive cough thirteen months after corticosteroid and immunosuppressant therapy. On the second admission, a chest CT scan detected the emergence of crazy-paving pattern in bilateral upper lobes. Bronchoalveolar lavage (BAL) fluid with milky appearance and a lung biopsy specimen revealed acellular periodic acid-schiff stain-positive bodies. The serum titer of anti-granulocyte macrophage colony stimulating factor (GM-CSF) antibodies was elevated on first admission and remained high on second admission. We thus diagnosed her as having autoimmune PAP. Reducing the dose of immunosuppressive agents and repeating the segmental BAL resulted in the improvement of her symptoms and radiological findings. Immunosuppressant therapy may trigger the onset of autoimmune PAP in a subset of patients with systemic sclerosis and/or sarcoidosis.

Serum YKL-40 is a reliable biomarker for pulmonary alveolar proteinosis.

BACKGROUND AND OBJECTIVE: Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by alveolar filling. YKL-40, a chitinase-like protein produced by macrophages and epithelial cells, is increased in patients with interstitial lung diseases. We aimed to evaluate the role of YKL-40 as a biomarker for PAP. METHODS: A total of 34 patients with autoimmune PAP and 50 healthy controls were studied. YKL-40 was measured by ELISA in serum and bronchoalveolar lavage fluid (BALF). Chitinase coding gene polymorphisms (CHI3L1-329 and -131) were detected by PCR and pyrosequencing. Correlations between serum YKL-40 levels and disease outcome were analysed. RESULTS: Baseline serum and BALF levels of YKL-40 were higher in PAP patients than in controls (286 ± 27 ng/mL vs 42 ± 4 ng/mL, P < 0.0001; 323 ± 36 ng/mL vs 3 ± 1 ng/mL, P < 0.0001, respectively). Serum YKL-40 levels correlated with diffusing capacity of the lung for carbon monoxide (DLCO ) at baseline (P = 0.002) and over time (P < 0.0001). Patients with disease progression had higher baseline serum YKL-40 levels than those who remained stable or improved (P < 0.0001). A baseline cut-off level of 300 ng/mL was predictive of disease progression (HR (hazard ratio): 7.875, P = 0.001). The presence of the G allele was associated with higher serum and BALF levels of YKL-40. CONCLUSION: YKL-40 is elevated in serum and BALF of PAP patients, and may be of clinical utility to predict outcome in PAP.

Update on Lysinuric Protein Intolerance, a Multi-faceted Disease Retrospective cohort analysis from birth to adulthood.

BACKGROUND: Lysinuric protein intolerance (LPI) is a rare metabolic disease resulting from recessive-inherited mutations in the SLC7A7 gene encoding the cationic amino-acids transporter subunit y+LAT1. The disease is characterised by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous ranging from infiltrative lung disease, kidney failure to auto-immune complications. This retrospective study of all cases treated at Necker Hospital (Paris, France) since 1977 describes LPI in both children and adults in order to improve therapeutic management. RESULTS: Sixteen patients diagnosed with LPI (12 males, 4 females, from 9 families) were followed for a mean of 11.4 years (min-max: 0.4-37.0 years). Presenting signs were failure to thrive (n = 9), gastrointestinal disorders (n = 2), cytopenia (n = 6), hyperammonemia (n = 10) with acute encephalopathy (n = 4) or developmental disability (n = 3), and proteinuria (n = 1). During follow-up, 5 patients presented with acute hyperammonemia, and 8 presented with developmental disability. Kidney disease was observed in all patients: tubulopathy (11/11), proteinuria (4/16) and kidney failure (7/16), which was more common in older patients (mean age of onset 17.7 years, standard deviation 5.33 years), with heterogeneous patterns including a lupus nephritis. We noticed a case of myocardial infarction in a 34-year-old adult. Failure to thrive and signs of haemophagocytic-lymphohistiocytosis were almost constant. Recurrent acute pancreatitis occurred in 2 patients. Ten patients developed an early lung disease. Six died at the mean age of 4 years from pulmonary alveolar proteinosis. This pulmonary involvement was significantly associated with death. Age-adjusted plasma lysine concentrations at diagnosis showed a trend toward increased values in patients with a severe disease course and premature death (Wilcoxon p = 0.08; logrank, p = 0.17). Age at diagnosis was a borderline predictor of overall survival (logrank, p = 0.16). CONCLUSIONS: As expected, early pulmonary involvement with alveolar proteinosis is frequent and severe, being associated with an increased risk of death. Kidney disease frequently occurs in older patients. Cardiovascular and pancreatic involvement has expanded the scope of complications. A borderline association between increased levels of plasma lysine and poorer outome is suggested. Greater efforts at prevention are warranted to optimise the long-term management in these patients.

Surfactant protein B: From biochemistry to its potential role as diagnostic and prognostic marker in heart failure.

Growing interest raised on circulating biomarkers of structural alveolar-capillary unit damage and very recent data support surfactant protein type B (SP-B) as the most promising candidate in this setting. With respect to other proteins proposed as possible markers of lung damage, SP-B has some unique qualities: it is critical for the assembly of pulmonary surfactant, making its lack incompatible with life; it has no other known site of synthesis except alveolar epithelial cells different from other surfactant proteins; and, it undergoes a proteolytic processing in a pulmonary-cell-specific manner. In the recent years circulating SP-B isoforms, mature or immature, have been demonstrated to be detectable in the circulation depending on the magnitude of the damage of alveolar capillary membrane. In the present review, we summarize the recent knowledge on SP-B regulation, function and we discuss its potential role as reliable biological marker of alveolar capillary membrane (dys)function in the context of heart failure.

MUC1 gene polymorphisms are associated with serum KL-6 levels and pulmonary dysfunction in pulmonary alveolar proteinosis.

BACKGROUND: KL-6, a human MUC1 mucin, is a sensitive biomarker for interstitial lung diseases including pulmonary alveolar proteinosis (PAP). A correlation between MUC1 gene single nucleotide polymorphism (SNP) rs4072037 genotype and serum KL-6 levels has been reported. This study was aimed at investigating the correlation between MUC1 SNP genotype, severity of disease and disease outcome in PAP. METHODS: Twenty four patients with PAP and 30 healthy volunteers were studied. MUC1 rs4072037 was detected by using a real-time polymerase chain reaction (RT-PCR). Genotyping was performed by pyrosequencing. KL-6 levels were measured in serum by Nanopia KL-6 assay (SEKISUI Diagnostics). RESULTS: The frequency of MUC1 rs4072037 alleles was significantly different between PAP patients and healthy volunteers (PAP, A/A 46%, A/G 54%, G/G 0%; healthy controls, A/A 30%, A/G 40%, G/G 30%; p = 0.013). Serum KL-6 levels were significantly higher in PAP patients than in controls (p < 0.0001), and significantly higher in PAP patients with A/A genotype than in those with A/G genotype (p = 0.007). Patients with A/A genotype had higher alveolar-arterial oxygen difference (A-aDO2) and lower DLco compared to those with A/G genotype (p = 0.027 and p = 0.012, respectively). Multivariate analysis, Kaplan-Meier analysis and C statistics showed that the rs4072037 A/A genotype was associated with higher rate of disease progression (HR: 5.557, p = 0.014). CONCLUSIONS: MUC1 rs4072037 A/A genotype is associated with more severe pulmonary dysfunction and a higher rate of disease progression in PAP patients.

Occupational inhalational exposure and serum GM-CSF autoantibody in pulmonary alveolar proteinosis.

OBJECTIVES: Although the serum granulocyte-macrophage colony stimulating factor autoantibody (GMAb) levels have been recognised as a diagnostic marker in primary pulmonary alveolar proteinosis (PAP), their role in PAP with occupational inhalational exposure (PAPo) remains unclear. METHODS: Forty-five consecutive patients with PAP were enrolled. Each patient with PAP was assessed for baseline clinical characteristics, chest high-resolution CT (HRCT), serum GMAb and occupational exposure. Fifty healthy controls were included to define normal ranges for GMAb levels. Ninety-seven hospital controls with other respiratory diseases were included to establish prevalence of a history of occupational inhalation exposure. RESULTS: According to the serum GMAb cut-off value of 2.39 µg/mL, 84.4% of the recruited patients with PAP had positive serum GMAb with a median level of 28.7 µg/mL, defined as autoimmune PAP, and the remaining 15.6% had negative serum GMAb with a median level of 0.16 µg/mL, defined as non-autoimmune PAP. Also, 34.2% of patients with autoimmune PAP had a history of occupational inhalational exposure, which was not significantly higher than that of hospital controls (34.2% vs 19.6%, p=0.072). Four patients with PAPo showed negative GMAb. Their arterial oxygen tension, pulmonary function parameters and chest HRCT features were significantly different when compared with patients with autoimmune PAP (p<0.05). These four non-autoimmune occupational lung disease cases culminated in 3 deaths and a lung transplant. CONCLUSIONS: A number of patients with PAP who may have occupational inhalational exposure and negative serum GMAb represent a high possibility of silicoproteinosis and very poor survival.

Pulmonary alveolar proteinosis.

The term pulmonary alveolar proteinosis (PAP) comprises a heterogeneous group of rare disorders characterized by abundant deposition of surfactant- and lipoproteins in the alveoli. The autoimmune form accounts for 90% of cases and is characterized by the presence of GM-CSF autoantibodies. Secondary PAP is associated with several underlying conditions, mainly hematologic malignancies, infections and inhalation exposure, and is GM-CSF antibody negative. Several conditions can mimic PAP, in particular the radiological findings: the crazy paving pattern on high resolution computed tomography (HRCT) is common also to infections, neoplasms, and other interstitial lung diseases. Bronchoalveolar lavage (BAL) typical findings and the detection of serum GM-CSF antibodies are usually sufficient for the diagnosis of PAP. Whole lung lavage (WLL) is still the gold standard for treatment of PAP and is followed by complete remission in about 50% of cases. Inhalative treatment with GM-CSF alone or in combination with WLL could represent the future approach for patients with autoimmune PAP refractory to WLL alone. The anti CD-20 antibody rituximab represents a further promising approach for autoimmune PAP. The treatment of secondary PAP should be focused on the underlying disease.

[Serum markers in patients with idiopathic pulmonary alveolar proteinosis].

OBJECTIVE: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by PAS positive lipoproteinaceous material accumulation in the pulmonary alveoli, of which autoimmune (APAP) or idiopathic type (IPAP) accounts for about 90%. Several serum markers were reported to be valuable in IPAP diagnosis. However, data in Chinese patients was not available. Here we analyzed the diagnostic value of serum markers in Chinese IPAP patients. METHODS: Thirty-one IPAP patients, 25 healthy volunteers and 15 patients with other lung diseases were enrolled in the study. Anti-GM-CSF antibody, SP-A, SP-D and YKL-40 levels of the serum samples were tested by ELISA method. LDH, CEA, arterial blood gas analysis and pulmonary function test results were collected and analyzed. Then the difference between the groups and the relationship between the 2 of the markers were tested. RESULTS: The level of serum anti-GM-CSF antibody, SP-A and SP-D [29.80(13.02-53.86) mg/L, (47 ± 30) and (77 ± 57) µg/L respectively] were higher in IPAP patients than in healthy volunteers and disease control patients (P < 0.05). IPAP and disease control groups had higher YKL-40 and LDH levels than the healthy control group (P < 0.05), but no statistical difference between the former 2 groups (P > 0.05). The CEA level of IPAP patients was higher than that of the disease control group (P < 0.05). The sensitivity and specificity of anti-GM-CSF antibody in IPAP diagnosis were both 100% at the cut-off value of 2.46 µg/mL. The LDH, CEA and SP-A levels correlated positively with P(A-a) O2 and negatively with TL(CO)%, PaO2 and SaO2 (P < 0.05). The SP-D level correlated negatively with SaO2 (P < 0.05), and positively with LDH and CEA level (P < 0.05), so did SP-A (P < 0.05). YKL-40 correlated positively with SP-A but not with other markers. Anti-GM-CSF antibody did not correlate with any of the markers. CONCLUSIONS: Serum anti-GM-CSF antibody testing showed high sensitivity and specificity for the diagnosis of APAP, though it did not correlate with disease severity. Serum LDH, CEA, SP-A and SP-D levels were elevated in IPAP patients and correlated well with disease severity. YKL-40 level was also higher in IPAP patients, but it could not be used as a disease severity marker.

CYFRA 21-1 as a disease severity marker for autoimmune pulmonary alveolar proteinosis.

BACKGROUND AND OBJECTIVE: Serum markers, including Krebs von den Lungen (KL-6), surfactant protein (SP)-D, SP-A and carcinoembryonic antigen (CEA), are reported to reflect autoimmune pulmonary alveolar proteinosis (APAP) disease severity. We evaluated serum CYFRA21-1 levels as a marker of APAP. METHODS: In addition to KL-6, SP-D and CEA, we prospectively measured serum CYFRA 21-1 levels in 48 patients with APAP, consecutively diagnosed between 2002 and 2010. Diagnostic usefulness of CYFRA 21-1 was determined from 68 patients with interstitial lung diseases by receiver operator characteristic curve analysis. We evaluated the association between these serum markers and other disease severity markers, including pulmonary function parameters, alveolar-arterial oxygen gradient, British Medical Research Council score reflecting shortness of breath, and disease severity score. CYFRA 21-1 localization in the lung was examined by immunohistochemistry. RESULTS: Receiver operator characteristic curve demonstrated that CYFRA 21-1 effectively identified APAP. Serum CYFRA 21-1 levels at diagnosis were significantly associated with the measured disease severity parameters. Following whole lung lavage (n = 10) and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation (n = 20), serum CYFRA 21-1 levels were significantly decreased. Responders (n = 11) to GM-CSF inhalation revealed significantly higher serum CYFRA 21-1 levels than non-responders (n = 9). Serum CYFRA 21-1 appeared to be a significant predictor of effectiveness of GM-CSF based on regression analysis. Immunohistochemistry showed that CYFRA 21-1 was localized on hyperplastic alveolar type II cells and lipoproteinaceous substances in alveoli. CONCLUSIONS: Serum CYFRA 21-1 is a sensitive and useful serum marker for diagnosis and evaluation of disease severity of APAP, and may predict the response to GM-CSF inhalation.