Extracavitary primary effusion lymphoma: clinical, morphological, phenotypic and cytogenetic characterization using nuclei enrichment technique.

AIMS: Primary effusion lymphoma (PEL) is a rare form of aggressive B-cell lymphoma, which typically manifests as malignant effusion in the body cavities. However, extracavitary solid variants are also described. The aim of this study was to investigate copy number aberrations in two cases of solid PEL at their first occurrences and relapse by applying a newly developed methodology of tumour nuclei enrichment. METHODS AND RESULTS: Using histological and genetic techniques, a novel protocol for tumour nuclei enrichment by flow sorting and array-comparative genomic hybridization, we characterized two cases of extracavitary PEL, one of which later relapsed as effusion. Both primary tumours were positive for HHV8 and EBV, confined to lymph nodes, and aberrantly expressed CD3, yet displaying clonal immunoglobulin gene rearrangements indicating B-cell origin. Cytogenetic characterization of primary tumours revealed modest number of aberrations, partially overlapping with previously reported affected loci. The effusional relapse in case 1 was cytogenetically related to the primary tumour but showed dramatic increase of chromosomal instability. CONCLUSIONS: We for the first time demonstrate a cytogenetic relationship between solid and effusional presentations of PEL. Moreover, we provide an indirect evidence of multiple malignant clones, which gave rise to clonally-related, yet karyotypically different relapsing lymphoma manifestations.

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

Clonal relationship of classical hodgkin lymphoma and its recurrences.

PURPOSE: We investigated whether the first and all subsequent manifestations of Hodgkin lymphoma (HL) in a patient are clonally related. EXPERIMENTAL DESIGN: We identified a collective of 20 patients with sometimes multiple HL recurrences. Relapses were classified as early, that is, within twelve months (eight events in seven patients) or as late, that is, later than one year after the previous neoplasm (24 events in 17 patients). Hodgkin and Reed-Sternberg cells were microdissected after CD30 staining using laser capture technique. Immunoglobulin heavy chain (IgH) gene fragment lengths were analyzed after DNA preamplification, applying consensus FR3 and J primers by ABI 310 Genetic Analyzer. Sequencing of the amplified IgH products was carried out by ABI 3130 and 3730XL Genetic Analyzer. Epstein-Barr virus (EBV) association was assessed by EBV early RNA and LMP1. RESULTS: Three cases with early relapses after a first HL diagnosis were clonally related to the initial tumor, whereas three of four patients with early relapses after a first or second relapse were not, which was accompanied by change of EBV association in one case. Six patients presenting with late relapses were clonally unrelated, which was accompanied by change of phenotype in two cases and change of EBV association in one case. Two samples from recurrent tumors of the same patient could be successfully sequenced. These two late relapses were clonally unrelated by IgH fragment length and sequencing analysis. CONCLUSIONS: Recurrent HL, especially those accompanied by an EBV-association switch or after a relapse, can represent an unrelated novel neoplasm. Our finding might play a role in clinical decision making.

Homozygosity for the c.917A→T (p.N306l) polymorphism in the EVER2/TMC8 gene of two sisters with epidermodysplasia verruciformis Lewandowsky-Lutz originally described by Wilhelm Lutz.

BACKGROUND: Epidermodysplasia verruciformis Lewandowsky-Lutz (EV) is a rare genodermatosis, characterised by development of numerous verrucous skin lesions caused by specific genotypes of human papillomaviruses belonging to the ß-papillomavirus genus. The EV loci were mapped to chromosome 2p21-p24 (EV2) and 17q25 (EV1). On chromosome 17, 2 adjacent related genes--EVER1/TMC6 and EVER2/TMC8--were identified. We reinvestigated 2 patients originally described by Wilhelm Lutz in 1946 with the aim to document the natural course of the disease and confirm his diagnosis. METHODS: PCR fragments specific for exons with short flanking intron sequences of EVER1/TMC6 and EVER2/TMC8 genes from patients' DNA were amplified using sequence information. The single-nucleotide polymorphism (SNP) rs7208422 was studied, using restriction fragment length polymorphism analysis. RESULTS: In the index patient, we identified a homozygous TT genotype in exon 8 of the EVER2/TMC8 gene (c.917A→T, p.N306I). The same mutation could thereafter be detected in her sister from paraffin-embedded skin. CONCLUSION: We have followed one of the first patients described with EV in Basel, Switzerland, in 1930 until today and demonstrated the TT genotype (SNP rs7208422) in the EVER2/TMC8 gene in this index patient and her sister. The results underline the possible relevance of SNP rs7208422 by influencing the susceptibility to ß-papillomaviruses and their oncogenic potential.

Combined analysis of specific KRAS mutation, BRAF and microsatellite instability identifies prognostic subgroups of sporadic and hereditary colorectal cancer.

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF(V600E) have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF(V600E) and specific KRAS mutation (Gly → Asp; G12D, Gly → Asp, G13D; Gly → Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF(V600E) was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild-type cancers (p = 0.038). With MSI, specific KRAS and BRAF(V600E) mutations, 3 distinct prognostic subgroups were observed in univariate (p = 0.006) and multivariable (p = 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF(V600E) mutation, (ii) KRAS/BRAF(V600E) wild-type or KRAS G13D mutations in MSS/MSI-L and (iii) MSI-H and KRAS G13D mutations. Moreover, none of the sporadic MSI-H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF(V600E) and MSI may identify sporadic CRC patients with poor clinical outcome.