SERUM ALVEOMUCIN IN PATIENTS WITH INTERSTITIAL LUNG DISEASES

See also IFUs for EIA kit

Medical research in co-operation with Institute of Pulmonology, Moscow, Russia
Polymorphic epithelial mucins are heavily glycolated glycoproteins of more than 300 kDa, expressed on apical cell surface of glandular epithelia. Mucin synthesized as a transmembrane molecule and there main fUction is participation in cell-cell adhesive interaction. Detection of certain mucins (CA 15.3, CA 125, CASA) has a very important role for screening and monitoring of epithelial neoplasmes. The new promising approach in this area is determination of serum mucin antigens (SSEA-1, KL-6, Alveomucin) in patients with pulmonary fibrosis (or interstitial lung diseases).

Interstitial lung diseases (ILD) includes a diverse group of acute and chronic disorders involving the lung parenchyma characterized by damage to normal epithelium, degeneration and necrosis. The main features of ILD are progressive dyspnea, bilateral interstitial shadows on chest radiograph and restrictive pulmonary dysfUction. Histologically the central feature of IPF are the marked loss of type 1 cells in the lung tissue and reepithelization of the epithelial surface with remaining type 2 alveolar cells, which are often hyperplastic and hypertrophic.. . It was shown in vitro that epithelial mucins are derived from regenerating type 2 pneumocytes in the lower respiratory tract. Elevated levels of serum mucins may reflect the whole burden of proliferating type 2 alveolar cell in deranged and fibrotic lungs.

Serum Alveomucin analysis. A sandwich immUoassay (K240) for detection of original glycolyl-sialylated glycoprotein (mucin) antigen Alveomucin was constructed using a monoclonal antibody 3E1.2 (Austin Research Institute, Australia) and affinity-purified polyclonal antibody G5 against total mucin antigen family. Using this assay we have quantified a serum mucin in all investigated patients

Monoclonal antibody 3E1.2 shows an unique specificity towards type II alveolocytes (see IHC picture below). This type of cells undergo dramatic hyperplastic growth during ILD onset and this may explain the hyperproduction of Alveomucin.

The role of serum Alveomucin as a screening test for ILD.

Serum levels off mucin Alveomucin were examined in patients with most common ILD forms - idiopathic pulmonary fibrosis (IPF) and chronic hypersensetivity pneumonitis (HP) and in other pulmonary (non-ILD) diseases. The preliminary study (1) included 14 patients with IPF (mean age 67+ 12 years), 14 with HP (mean age 44+ 13 years), 40 with bronchial asthma (BA)( 46+ 9years), 18 with chronic obstructive pulmonary disease (COPD)(66+ 8 years), 20 with pneumonia (42 + 11 years) and 52 healthy volUteers (30 + 4 years).

ILD diagnosis was confirmed by hystological examination of open-lung biopsy specimens and by transbronchial specimens and for HP patients there was serologic evidence of precipitating antibodies against offending antigens.

An arbitrary cut-off 75 Uits/ml (U/ml) was set as mean + 2SD (n=52) of random population. Most of ILD patients have shown an elevation of serum mucin Alveomucin up to 595 U/ml (mean 226 + 112 U/ml, n=24). Serum Alveomucin was not elevated in acute pneumonia (49 + 31 U/ml), COPD (78 +31 U/ml), BA (69 + 33 U/ml), also moderate elevation of serum Alveomucin levels (up to 169 U/ml) was observed in severe bronchial asthma, especially in children. The elevated serum mucin Alveomucin was detected in all studied forms of ILD.

Figure 1. Serum levels of Alveomucin in different pulmonary diseases.

In all of examined patients of had serum levels of Alveomucin more than 100 U/ml, so calculated the sensitivity, specificity and diagnostic accuracy of mucin levels for two cut-off points: 75 and 100 U/ml. Specificity and diagnostic accuracy of Alveomucin increased with the use of cut-off level of 100 U/ml without decreasing the sensitivity of test (2).

Sensitivity, specificity and diagnostic accuracy of Alveomucin for ILD.

Alveomucin > 75 U/ml Alveomucin > 100 U/ml

Sensitivity, % 100 100

Sensitivity, % 76 91

Diagnostic accuracy, % 81 93

In conclusion, determination of serum mucin Alveomucin may serve as a preliminary diagnostic marker of some forms of ILD.

Serum mucin Alveomucin as a marker of activity and severity of ILD

Usually disease activity is assessed from tissue obtained by open or transbronchial lung biopsy, but it not practical to perform these frequently and due to non-uniform distribution of histological changes throughout the lung small samples may be non-representative. Many attempts have been made to find markers of disease in ILD which may define patients at risk for progressive and irreversible lung damage.

The degree of serum mucin elevation in ILD patients was correlated with severity of clinical symptoms, degree of restrictive changes in pulmonary function tests: parameters of gas exchange and polymorphic neutrophils (PMN) count in bronchoalveolar lavage (BAL) fluid (only for IPF) (3).

Correlation between serum levels of Alveomucin and functional parameters in ILD patients (n=28).

Parameter R P

DLCO % pred - 0,64 < 0,001

DLCO/Va % pred. - 0,52 0,005

FVC % pred. - 0,46 0,014

FEV1 % pred - 0,57 0,002

TLC % pred. - 0,46 0,015

RV % pred - 0,30 0,132

Neutrophils BAL, % (for IPF) 0,87 < 0,001

РаО2, mm Hg - 0,65 < 0,001

Р(А-а)О2, mm Hg - 0,59 < 0,001

Correlation between serum Alveomucin (3EG5) and a) DLCO and b) P(A-a)O2

Thus it may be concluded that serum levels of Alveomucin may reflect severity and activity of ILD.

The use of successive measurements of serum mucin antigen Alveomucin for monitoring of immUosupressive therapy in patients with ILD

Serum levels of Alveomucin are significantly elevated in chronic ILD and reflect the severity of ILD: a degree of serum mucin elevation in these patients was correlated with changes in pulmonary function tests (FVC, DLCO) and gas exchange abnormalities. As these variables are traditionally used for estimation of therapeutic response in ILD patients the usefulness of Alveomucin in prediction of therapeutic response was evaluated

We investigated the relationship between functional response to the anti-inflammatory therapy and changes in serum Alveomucin in ILD patients (4).

We have included in our study 16 patients with chronic pneumonitis: 9 with idiopathic pulmonary fibrosis (IPF) (mean age 68 + 11 years) and 7 with hypersensitivity pneumonitis (HP) (mean age 42 + 14 years). All patients were treated with immunosuppressive therapy (prednisolone- all, azathioprine- 1, cycloposphamide- 1, penicillamine- 1) and their functional variables were assessed before and after 6 months of therapy.

According changes in pulmonary function tests during 6-months of steroid treatment all patients were divided into two groups: responders (15 % increase in FVC, 20 % increase in DLCO and reduction in the PA-aO2 of 5 mm Hg) and nonresponders (worsening or nonsignificant changes in these variables).

Figure. Serum mucin Alveomucin levels before and after of 6th month immUnosupressive therapy in responders and nonresponders groups of ILD patients.

In responders (n= 7) serum levels of Alveomucin fell after treatment from 230 + 97 to 88 + 40 Uits/ml (p< 0,01). In nonresponders serum Alveomucin were not different before and after therapy: 249 + 153 and 247 + 170 Uits/m (p < 0,05) Two nonresponding patients showed significant decrease in Alveomucin (all- with HP), so the accuracy of changes in serum Alveomucin to reflect response to therapy was 88 %. There was a significant difference between the posttreatment levels of Alveomucin of the two groups of patients (p= 0,03).

The results obtained lead to the conclusion that successive measurements of serum mucin antigen Alveomucin may be used for predicting the response to the immUosuppressive therapy in ILD patients.

Comparison of different serum mucin tests and Alveomucin as the markers of severity and activity of IPF

Detection of certain mucins are traditionally used for screening and monitoring of epithelial neoplasmes but the same mucins may be elevated in patients with ILD We compared the serum levels of commercially available mucins CA125, CA15-3, MCA, MSA andAlveomucin specially designed for ILD in IPF patients and to evaluate the relationship between these mucins and markers of severity and activity of IPF

Serum levels of mucin markers were examined in 17 patients with confirmed IPF (age: 57+ 10 years; FVC: 81+ 32 % pred.; DLCO: 65+ 26 % pred.; P(A-a)O2: 29+ 13 mm Hg) (5).

All examined mucins were elevated in IPF, also the positivity rates were different.

Mucin	Mean + SD	Range	Cut-off level	positivity (n/n)	positivity (%)
Alveomucin	217 + 129	108- 595	75	17/17	100
MSA	203 + 221	22- 600	30	16/17	94,1
CA15-3	90 + 71	22- 210	28	15/17	88,2
MCA	21+ 13	6- 50	15	11/16	68,8
CA125	69 + 136	3- 540	35	6/17	35,3

All mucin markers showed significant correlation with some of indices of severity and activity of IPF. The best correlations were observed for mucin Alveomucin.

	Alveomucin	MSA	CA-15.3	MCA	CA-125
DLCO	-0,51 (p=0,03)	-0,55 (p=0,02)	-0,39 (p=0,12)	-0,33 (p=0,21)	-0,44 (p=0,08)
FVC	-0,52 (p=0,03)	-0,49 (p=0,07)	-0,58 (p=0,01)	-0,36 (p=0,17)	-0,38 (p=0,13)
BAL neut	0,79 (p<0,01)	0,61 (p=0,01)	0,46 (p=0,05)	0,52 (p=0,04)	0,50 (p=0,04)

These data show that although the serum levels of different mucins (CA-15.3, CA-125, MSA, MCA) are elevated in IPF patients mucin Alveomucin is a more valuable text in these patients.

References:

1. Avdeeva O., Lebedin Y., Avdeev S., Chukanov S., Chuchalin A.G. Serum glycolyl-sialylated glycoprotein antigen Alveomucin as a laboratory marker for interstitial lung disease. Sarcoidosis 1997; 14: 37S.
2. Avdeeva O., Lebedin Y., Avdeev S., Chukanov S., Chuchalin A.G., Cherniack A. Serum glycolyl-sialylated mucin antigen Alveomucin in patients with interstitial lung disease. Eur.Respir.J. 1997; 10: 262S.
3. Chuchalin A.G., Avdeeva O., Lebedin Y., Avdeev S., Chukanov S. An elevated level of glycolyl-sialyated mucin antigen Alveomucin in patients with interstitial lung diseases. Am.J.Respir.Crit.Care Med. 1998; 157(Suppl.): A39.
4. Avdeeva O., Lebedin Y., Avdeev S.N., Cherniack A., Chuchalin A. Successive measurements of serum mucin antigen Alveomucin before and after immUosuppressive therapy in patients with ILD. Eur.Respir.J. 1998; 11: 165S.
5. Avdeeva, Y. Lebedin, S. Avdeev, A. Chuchalin. Role of serum mucins as markers of severity and activity of idiopathic pulmonary fibrosis (IPF). Eur.Respir.J. 1999; 12: (in press).
6. Yokoyama A., Kohno N., Hamada H., Sakatani M., Ueda E., Kondo K., Hirasawa Y., Hiwada K. Circulating KL-6 predicts the outcome of rapidly progressive idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 1998;158:1680- 1684.
7. Yokoyama A., Kohno N., Kondo K., Ueda S., Hirasawa Y., Watanabe K., Takada Y., Hiwada K. Comparative evaluation of sialylated carbohydrate antigens, KL-6, CA19-9 and SLX as serum markers for interstitial pneumonia. Respirology 1998;3:199- 202.
8. Satoh H., Kamma H., Ogata T., Yano H., Ohtsuka M., Hasegawa S. Clinical significance of serum levels of a carbohydrate antigen, syalyl SSEA-1, in patients with fibrosing lung disease. Am.Rev.Respir.Dis. 1991; 144: 1177- 1181.
9. Norum L.F., Varaas T., Kierulf B., Nustad K. Carcinoma-associated MUC1 detected by immUoradiometric assays. Tumour Biol 1998; 19 Suppl 1: 134- 146
10. Smart Y.C., Stewart J.F., Bartlett L.D., Brien J.H., Forbes J.F., Burton R.C. Mammary serum antigen (MSA) in advanced breast cancer. Breast Cancer Res Treat 1990; 16: 23- 28