Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA).

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjogren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P < 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.

Higher risk of mismatch repair-deficient colorectal cancer in alpha(1)-antitrypsin deficiency carriers and cigarette smokers.

Microsatellite instability (MSI) is a genomic alteration observed in 15-30% of colorectal cancer (CRC). Two MSI phenotypes have been defined for CRC: MSI-H is characterized by MSI at > or =30% of the examined loci and MSI-L by MSI at 1-30% of the loci. An absence of MSI at any examined loci has been defined as a microsatellite stable (MSS) phenotype. Current data suggest the majority of MSI tumors are the result of defective DNA mismatch repair (MMR). In this study, we have determined the alpha(1)-antitrypsin deficiency carrier (alpha(1)ATD-ht) status of 161 CRC patients whose MSI phenotype and protein expression states had previously been determined. Cases were selected to enrich a larger number of MSI-H cases. Among 51 CRC patients with MSI-H tumors, the alpha(1)ATD-ht rate was 21.6%; among 110 patients with MSI-L/MSS tumors, the rate was 9.1% (MSI-H vs MSI-L/MSS, P = 0.02); and among the 191 population-based controls the alpha(1)ATD-ht rate was 9.4% (MSI-H vs controls, P = 0.02). The estimated relative risk of having MSI-H CRC among alpha(1)ATD-ht was 3.1 after adjusting for age, gender, and smoking history. The risk of having MSI-H CRC among current and past smokers was 6.6 and 2.7, respectively. Patients who were alpha(1)ATD-ht and smoked had a 20-fold increased risk of developing an MSI-H CRC compared to nonsmokers who were homozygous normal at the alpha(1)ATD locus. Our findings suggest an etiologic link between alpha(1)ATD alleles and development of CRC with defective MMR, and a synergistic effect between smoking and alpha(1)ATD allele in the development of MSI-H CRC.