Gastrointestinal stromal tumors - Summary of mutational status of the primary/secondary KIT/PDGFRA mutations, BRAF mutations and SDH defects.

The most important findings revealing pathogenesis, molecular characteristics, genotyping and targeted therapy of gastrointestinal stromal tumors (GISTs) are activated oncogenic mutations in KIT and PDGFRA genes. Imatinib mesylate (IM), which inhibits both KIT and PDGFRA receptors, significantly improved treatment of advanced (metastatic, recurrent, and/or inoperable) GISTs. However, in a significant number of patients the treatment fails due to the primary or secondary resistance to targeted therapy. Most common cause of secondary resistance is a presence of secondary mutations. Approximately 15% of adult patients with GISTs are negative for mutations in KIT or PDGFRA genes. These so-called wild-type GISTs appear to be characterized by other oncogenetic drivers, including mutations in BRAF, RAS, NF1 genes, and subunits of succinate dehydrogenase (SDH) complex. In the present study we investigated 261 tumour specimens from 239 patients with GIST. Primary mutations were detected in 82 % tumor specimens. 66 of them were in KIT, and 16 % in PDGFRA genes. Remaining 18 % were KIT/PDGFRA wild-type. Secondary KIT mutations were detected in 10 from 133 (7 %) patients treated with IM. We examined secondary KIT mutations in exons 13 and 17 and secondary PDGFRA mutation in exon 18 in sixteen progressive tumors and/or metastasis (from overall 22 samples). We identified BRAF V600E point mutation in 4 % of KIT/PDGFRA wild-type GIST patients. Moreover, we analysed SDH complex mutations in 4 younger patients (15, 33, 37, and 45 years old) from 44 patients without KIT, PDGFRA, and BRAF mutations. Two patients (a 37-year old man, and a 33-year old woman) had defects of the SDH complex. Our findings of mutational status of the primary and secondary KIT/PDGFRA mutations in patients with GIST confirm mechanisms of primary and secondary resistance, and also intralesional and interlesional heterogeneity of secondary mutations within and between progressive lesions. Moreover, detection of V600E BRAF mutation and defects of SDH complex in KIT/PDGFRA wild-type GISTs confirm their activation and allow for a selection of targeted therapy.

Detection and clinical significance of bone marrow involvement in patients with rhabdomyosarcoma.

Childhood rhabdomyosarcoma (RMS) has two major histological subtypes: alveolar (aRMS) and embryonal. The aim of the study was to monitor minimal disseminated disease (MDD) using real-time quantitative reverse-transcription PCR (RQ-RT-PCR) of the PAX3-FKHR, PAX7-FKHR fusion genes and myoD1 gene. We prepared an assay using RQ-RT-PCR for a quantitative assessment of MDD in aRMS by using hydrolysis probe for quantification of PAX3-FKHR, PAX7-FKHR and myoD1 genes and beta-2-microglobulin housekeeping gene. Primary tumor samples (44), samples of local recurrences (26) from 48 patients with aRMS were examined by nested RT-PCR and RQ-RT-PCR techniques. Additionally, bone marrow samples (115), peripheral blood progenitor cell samples (27), and peripheral blood samples (25) from 33 aRMS patients were tested. PAX3/7-FKHR and myoD1 transcripts proved to be a sensitive tool for detection of MDD in RMS. We were able to identify 15/25 patients with bone marrow (BM) involvement at the time of presentation using RQ-RT-PCR. We analyzed PAX3-FKHR or PAX7-FKHR expression during the course of the disease. The RQ-RT-PCR results correlated well with nested RT-PCR results (p < 0.0001). The presence of metastases is the most adverse prognostic factor in RMS, and bone marrow is a frequent site of the tumor dissemination in RMS, especially in aRMS. Our results detecting the fusion transcripts or myoD1 transcript in the BM or peripheral blood suggest that patients with positive findings are at high risk of the tumor progression.

Relative quantification of ERBB2 mRNA in invasive duct carcinoma of the breast: correlation with ERBB-2 protein expression and ERBB2 gene copy number.

The option to treat patients suffering from ERBB-2 protein-positive invasive duct carcinomas of the breast (IDC) with Herceptin requires a precise determination of the ERBB2 status. The aim of the study was to evaluate the ERBB2 mRNA level, placing emphasis on cases with discordant findings between ERBB-2 protein expression (IHC) and a copy number of the ERBB2 gene (FISH). Thirty-nine IDCs (21 cases IHC and FISH concordant, 15 cases moderately discordant, 3 cases markedly discordant) were investigated. ERBB2 mRNA expression was determined using quantitative real-time RT-PCR (Q-RT-PCR). IDCs with negative ERBB-2 protein and without ERBB2 gene amplification had a low ERBB2 mRNA level. Cases with 3+ overexpression of the protein and with strong gene amplification (> 10 copies/tumor cell) had a significantly increased expression of ERBB2 mRNA. In 13 of 15 IDCs with moderate discrepancies (up to 10 copies of the gene per one tumor cell/negative ERBB-2 protein; without amplification/2+ protein) mRNA was low, comparable to that in cases with negative ERBB-2 protein and without ERBB2 gene amplification. In three cases with markedly discordant findings (the gene amplified/protein negative--one case; protein 3+/no amplification--2 cases), Q-RT-PCR results were within a "normal" limit. Ineffective gene amplification and protein accumulation are suggested explanations. Q-RT-PCR revealed two cases with highly expressed ERBB2 mRNA and discordant FISH and/or IHC findings. Increased effectiveness of transcription (protein 2+/high mRNA/without the gene amplification), and combined dysregulation (protein negative/high mRNA/no amplification) are possible causes of these findings. Q-RT-PCR appears useful in clarifying borderline or discrepant IHC and FISH findings.