Molecular characterization of an Italian series of sporadic GISTs.

PURPOSE: Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. Most (80 %) contain activating mutations in the KIT receptor tyrosine kinase, roughly 10 % in platelet-derived growth factor receptor-alpha (PDGFRA). In a small subset, BRAF mutations are an alternative molecular pathway. GISTs respond well to imatinib, but low response is seen in patients with wild-type KIT or PDGFRA. Resistance has also been reported as a result of mutations in downstream effectors such as BRAF. METHODS: We provide here a molecular characterization of a series of primary GISTs from Italian patients. Of 121 GIST cases diagnosed between 2000 and 2012, 83 were evaluated by PCR amplification and direct sequencing for mutations in KIT exons 8, 9, 11, 13, and 17, PDGFRA exons 12, 14, and 18, and BRAF exon 15. Eighty-one GISTs also underwent K-RAS testing. RESULTS: Sixty-four GISTs were positive: 55 had mutations in KIT and 9 in PDGFRA; 16 patients were mutation negative. Three samples came from NF1 patients and were KIT- and PDGFRA negative. Overall, we identified six novel mutations in KIT (p.K550_M552delinsL, p.Q556_W557delinsG p.Q556_G575del, p.W557_V559delinsQ p.P573_R588dup, p.G592_K593dup) and one novel mutation in PDGFRA (p.D842_N848delinsVDV), thus contributing to widening the spectrum of known mutations in GIST tumors and confirming the most frequently altered regions underlying GIST development. CONCLUSIONS: Among the 64 KIT- and PDGFRA-positive sporadic patients in our series, no BRAF or KRAS mutations were identified, suggesting that co-occurrence of these mutations is likely to be rare in the northwestern Italian population and not a frequent cause of primary resistance to imatinib in KIT-positive GIST patients.

The Genoa experience of prenatal diagnosis in NF1.

Type 1 neurofibromatosis (NF1) is an autosomal dominant disorder with an incidence of about 1 in 3500 live births. Symptoms are highly variable from a few cafè-au-lait spots and axillary freckling to plexiform neurofibromas, optic gliomas, pseudarthrosis, and malignancy. Since disease causing mutations are dispersed throughout the gene, prenatal diagnosis is usually performed in familial cases by linkage analysis and rarely by direct characterization of the mutation. We have characterized 48 families and have performed four prenatal diagnoses. In three cases, the linkage analysis was carried out using informative markers. A direct approach using the protein truncation test (PTT) and sequencing was performed in one case in which a R1947X mutation was identified. The extreme variability of the phenotypic expression of the NF1 gene makes reproductive decisions in NF1 families very difficult, as molecular diagnosis cannot predict clinical expression of the disease. The psychological management of the couple is therefore difficult. In two of the three examined families the reproductive choices were not influenced by the specific manifestations of the disease in that family.

Screening of neurofibromatosis type 1 gene: identification of a large deletion and of an intronic variant.

Neurofibromatosis type 1 of von Recklinghausen is a common autosomal dominant disorder, characterized by peripheral neurofibromas, café-au-lait spots and Lisch nodules of the iris. The high mutation rate at the neurofibromatosis type 1 locus results in a wide range of molecular abnormalities. We have screened seven different exons of the neurofibromatosis type 1 gene, including those codifying for the GAP-related domain, using the RNA-Single Strand Conformation Polymorphism (RNA-SSCP) method in a series of 59 neurofibromatosis type 1 patients. We have also analyzed four intragenic repeats and one RFLP to detect hemizygosity and evaluate informativeness in at-risk families. One deletion and a new intronic normal variant have been detected. Thus the majority of Neurofibromatosis type 1 chromosomes have not been characterized, confirming difficulty in providing proper genetic counselling in neurofibromatosis type 1 families, even following extensive DNA analysis.

Expression and genomic configuration of GM-CSF, IL-3, M-CSF receptor (C-FMS), early growth response gene-1 (EGR-1) and M-CSF genes in primary myelodysplastic syndromes.

Peripheral blood mononuclear cells from seventeen patients with primary myelodysplastic syndromes (MDS) in advanced stage were enriched for blasts and tested for (1) karyotype, (2) genomic configuration and (3) expression of IL-3, GM-CSF, FMS and EGR-1 genes which are all located on the long arm of chromosome 5. The expression of the M-CSF gene, that has been recently reassigned to the short arm of chromosome 1 (lp), was also investigated. Aims of the study were to (1) assess the potential role of the expression of these genes in the maintenance and expansion of the neoplastic clones and (2) search for constitutional losses or rearrangements of one allele followed by a deletion of the second allele of the same genes in the leukemic cells. The latter issue was investigated by comparing, in 8 cases, constitutive DNA from skin fibroblasts with leukemic DNA. Eleven of the 17 patients had abnormal karyotypes. The M-CSF gene was expressed in 6 cases and the FMS and the EGR-1 genes were expressed in 2 of the latter cases. An autocrine mechanism of growth could be hypothesized only for the 2 patients whose cells expressed both the M-CSF and FMS genes. No germline changes or rearrangements were observed in any of the genes studied. Thus, deregulation of genes encoding for certain hemopoietic growth factors or receptors does not seem to represent a major mechanism of MDS progression.

Detection of a neurofibromatosis type I (NF1) homologous sequence by PCR: implications for the diagnosis and screening of genetic diseases.

The neurofibromatosis type I (NF1) gene was extensively screened for mutations using single strand conformation polymorphism (SSCP) technology. During the analysis of the NF1 GAP-related domain, electrophoretically abnormal fragments were detected. Direct sequencing of these fragments allowed us to identify the presence of a NF1 highly homologous sequence (NF1HHS). A detailed analysis of a hybrid panel located this sequence on chromosome 15q24-->qter. An accurate search through several data banks demonstrated that this sequence is a new NF1 homologue. This report shows how it is possible to find homologous sequences at random, and subsequently to make wrong interpretations.

Linkage studies in Italian families with familial adenomatous polyposis.

Linkage analysis was performed on 188 subjects belonging to 18 Italian families segregating for familial adenomatous polyposis (FAP) using 7 polymorphic markers (5 restriction fragment length and 2 dinucleotide repeat polymorphisms) mapping in 5q21. A two-point linkage analysis performed with the LINKAGE program gave significant lod scores (> 3) between the Pi227, C11p11, YN5.64, YN5.48 probes and the disease, whereas the ECB27, CB83 and EF5.44 markers showed lower lod scores. Some 11 recombination events were identified from the analysis of 101 meioses. The best map that we could determine confirmed that reported in previous studies. The location of the new marker, CB83, lying between YN5.64 and YN5.48, remains imprecise. No genetic heterogeneity was detected, with all the families showing linkage for at least one of the probes. One 34-year-old individual having an affected haplotype was however classified as healthy after clinical examinations. The results confirm the applicability of the linkage approach for presymptomatic diagnosis of FAP.

Early and long term follow-up with minisatellite probes in bone marrow transplanted patients.

Twenty-five patients who received bone marrow transplantation (BMT) for chronic granulocytic leukemia (CGL), acute leukemia and severe aplastic anemia were studied before and after BMT in order to document and characterize the events following transplantation. DNA analysis was performed using minisatellite probes, which give rise to extremely polymorphic Southern blot band patterns specific to each individual and are regarded as "genetic fingerprint." Sensitivity studies using a mixture of donor and recipient cells could distinguish the presence of 1% of cells from one individual. Blood specimens were obtained from donor recipient before BMT and at days 10, 30, 90, and 270 after BMT. Karyotype analysis was also performed in CGL patients at the same time of DNA analysis. Engraftment was identified by DNA analysis as early as 10 days posttransplant and correlated with cytogenetic findings. This confirmed that a single hybridization filter is informative in 100% of patients and is easily applicable for early and long term studies of chimerism in BM transplanted patients.

Molecular analysis of Philadelphia-negative myeloproliferative syndromes with i(17q).

We report two cases of myeloproliferative syndromes in which the only karyotypic abnormality was an isochromosome of the long arm of chromosome 17. Because i(17q) is a nonrandom structural aberration found in nearly 12% of cases of Philadelphia (Ph)-positive chronic myelogenous leukemia (CML), we carried out a molecular analysis of the breakpoint cluster region (bcr) to verify the presence of genomic rearrangements characteristic of CML. The interest of the study was strengthened by the fact that i(17q) is frequently seen in CML and by recent reports showing that genomic changes of c-abl and bcr genes can be present even in the absence of a Ph chromosome. One of the two patients showed the presence of a rearranged fragment within the bcr, suggesting a Ph-positive CML diagnosis.

Translocation t(9;9)(p13;q34) in Philadelphia-negative chronic myeloid leukemia with breakpoint cluster region rearrangement.

Chromosome analysis showed a t(9;9)(p13;q34) in a patient with chronic myeloid leukemia (CML) without a Philadelphia (Ph) chromosome in all examined cells. Southern blot analysis of leukocyte DNA revealed rearrangement of breakpoint cluster region (bcr) within the 5.8-kb bcr sequences as in Ph-positive CML patients. The findings confirm that the 9q34 and 22q11 bands are always involved in CML independent of the chromosomal evidence. It is suggested that Ph-negative bcr-positive CML may have variant translocations, as in the case of the t(9;9) reported here.

Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia.

Leukemic cells with double minute (DM) chromosomes from an ANLL(M1) patient were found to carry 10-15 fold amplified c-myc sequences. The linked pvt-1-like locus was amplified at the same level, suggesting that the c-myc amplicon is at least 300 kb in size.

Involvement of chromosomal region 9q34 in a case of variant Ph1 translocation t(22;22).

In a patient with chronic myelocytic leukemia chromosome analysis showed a translocation (22;22) (q13;q11). Chromosomes 9 were apparently not involved. Using somatic cell hybrids and a v-abl probe, we demonstrated the translocation of c-abl sequences from chromosome 9 to chromosome 22q-. This confirms the hypothesis that the translocation of c-abl oncogene is essential for the development of Ph1 positive CML.