[Lynch Syndrome -  the Pathologist's Diagnosis]. / Diagnóza Lynchova syndromu od patologa.

Lynch syndrome (formerly known as hereditary non-polyposis colorectal cancer) is the most com-mon hereditary colorectal cancer syndrome. The syndrome is caused by a germline mutation of one of the mismatch repair (MMR) genes responsible for DNA replication error repair. Impaired function of the proteins encoded by these genes leads to microsatellite instability (MSI), which is associated with increased incidence of neoplasms: mainly colorectal cancer. According to recent estimates, up to 5% of all colorectal cancers are associated with Lynch syndrome. Due to this relatively high frequency, familial occurence, absence of premorbid phenotype, and development of malignant tumors at a reproductive age, a correct diagnosis is important not only from an ethical but also from an economical point of view. Unfortunately, clinical means of diagnosis, namely, the revised Bethesda guidelines designed to detect patients suitable for genetic testing for Lynch syndrome, lack sufficient sensitivity. The methods associated with modern pathology are more sensitive than the clinical criteria used to detect patients suspected of having Lynch syndrome. Pathological diagnostics are based on direct or indirect detection of MSI. Indirect methods include analysis of morphological signs associated with MSI in histological samples from colorectal carcinoma patients and immunohistochemical investigation of MMR protein expression. To rule out sporadic cases caused by epigenetic inactivation of an MMR gene, molecular genetic investigation of the BRAF gene and methylation analysis of the MLH1 promoter are performed during diagnostic workup. A suspicion of Lynch syndrome based on the results of the methods mentioned above should be proven by detection of a germline mutation in an MMR gene in peripheral blood leukocytes.

Utility of immunohistochemical investigation of SDHB and molecular genetic analysis of SDH genes in the differential diagnosis of mesenchymal tumors of GIT.

Loss of expression of beta subunit of succinate dehydrogenase (SDHB) was proved to be present in a subgroup of KIT/PDGFRA wt gastrointestinal stromal tumors (GISTs). To evaluate possible diagnostic utility of SDHB immunohistochemistry in the differential diagnostics of mesenchymal tumors of gastrointestinal tract (GIT), 11 cases of KIT/PDGFRA wt GISTs, 12 gastric schwannomas (GSs), 20 solitary fibrous tumors (SFTs), 4 leiomyomas (LMs), 16 leiomyosarcomas (LMSs), 5 synovial sarcomas (SSs), 3 endometrioid stromal sarcomas (ESSs), and 1 ileal inflammatory myofibroblastic tumor (IMT) were investigated for SDHB immunoexpression together with molecular genetic analysis of genes encoding succinate dehydrogenase (SDH). Three recent cases of KIT/PDGFRA mutant GISTs were used as controls. Among the 11 KIT/PDGFRA wt GISTs, 6 expressed SDHB, 1 of them harboring a sequence change of SDHD. All SDHB-negative cases were SDHB-D wt. In 1 of the control GIST cases molecular genetic analysis revealed an SDHD sequence change in addition to a mutation in KIT exon 11. No SFT was truly SDHB-negative, but in 2 of them the staining was impossible to analyze. Furthermore, 1 SFT carried an SDHB and another 1 SDHD sequence change. All GSs, LMs, LMSs, SSs, ESSs, and IMT were SDHB-positive or non-analyzable, and SDHB-D wt. Additional factors may play a role in regulating expression of SDHB. Furthermore, SDHB immunohistochemistry alone may be misleading in excluding tumors other than GIST (especially SFT) in the differential diagnosis of KIT/PDGFRA wt mesenchymal tumors of GIT.

Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis.

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.

Comparison of specificity and sensitivity of immunochemical and molecular techniques for reliable detection of Erwinia amylovora.

Erwinia amylovora [(BURRILL) WINSLOW et al.] (Ea), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IFAS and ELISA. Polyclonal antibodies of Neogen Europe Ltd. were used for IFAS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using the BIOLOG system and an immature pear fruit assay, identities of all Ea strains were confirmed as the fire blight bacterium. In assays of pure Ea cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 10(6)-10(5) and 10(5)-10(4) CFU/mL, respectively. When saprophytic bacteria associated with Ea in plant samples were tested as potentially cross-reacting bacteria, PTA-ELISA and IFAS gave 20 and 14 % cross-reactions, respectively. In plant samples, the presence of Ea was more reliably detected by IFAS (at a dilution of 1 : 1000) than by PTA-ELISA (to dilution 1 : 100). The capacity to detect Ea might be increased using an optimized PCR, but for PCR prepared from infected plant samples it was necessary to use the bacterial DNA isolated with a DNeasy Plant Mini Kit (Qiagen). In this case the PCR was sensitive to a concentration of 10(5) CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used.

Reliability of diagnostic techniques for Erwinia amylovora, the causative agent of fire blight disease.

A total of 20 putative strains of Erwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding to A620 = 0.1, while at A620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection of E. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain of E. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain of E. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 degrees C) for these primers was increased to 55 degrees C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification system Biolog those 3 strains were identified as Pantoea dispersa (1 strain) and Pantoea agglomerans (2 strains). The strains are supposed to be white variants of the species P. dispersa and P. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.