Evidence of Genetic Instability in Tumors and Normal Nearby Tissues

We have analyzed the sequence heterogeneity of the transcripts of the human HPRT and G6PD single copy genes that are not considered tumor markers. Analyses have been performed on different colon cancers and on the nearby histologically normal tissues of two male patients. Several copies of each cDNA, which were produced by cloning the RT-PCR-amplified fragments of the specific mRNA, have been sequenced. Similar analyses have been performed on blood samples of two ostensibly healthy males as reference controls. The sequence heterogeneity of the HPRT and G6PD genes was also determined on DNA from tumor tissues. The employed analytical approach revealed the presence of low-frequency mutations not detectable by other procedures. The results show that genetic heterogeneity is detectable in HPRT and G6PD transcripts in both tumors and nearby healthy tissues of the two studied colon tumors. Similar frequencies of mutations are observed in patient genomic DNA, indicating that mutations have a somatic origin. HPRT transcripts show genetic heterogeneity also in healthy individuals, in agreement with previous results on human T-cells, while G6PD transcript heterogeneity is a characteristic of the patient tissues. Interestingly, data on TP53 show little, if any, heterogeneity in the same tissues. CONCLUSIONS/SIGNIFICANCE: These findings show that genetic heterogeneity is a peculiarity not only of cancer cells but also of the normal tissue where a tumor arises

La maculatura clorotico rugginosa del ciliegio: Studi su funghi e micovirus come possibili agenti eziologici. Evidenza di endofitosi tra ciliegio e Taphrina wiesneri

Il lavoro di questa tesi è parte di un progetto di ricerca finanziato dal Ministero delle Politiche Agricole per lo studio della recente malattia delle macchie clorotico-rugginose del ciliegio (CCRS). Una patologia che si propaga rapidamente tra i ciliegi di uno stesso frutteto con effetti letali. Studi precedenti indicavano il coinvolgimento nella patologia sia di un fungo sia di micovirus. I risultati della mia tesi mostrano che il micovirus del genere Chrysovirus si rivela geneticamente solo nelle aree sintomatiche della foglia e non nelle aree asintomatiche circostanti né nelle nervature. Il virus si osserva ad alta concentrazione già in aree con sintomi molto precoci sia come genoma sia come proteina capsidica identificata per analisi di immunorivelazione mediante anticorpi prodotti con un costrutto genetico di antigeni da me progettato. Si è così accertato che la presenza del micovirus è un evento molto precoce e che l’effetto letale non è probabilmente dovuto a propagazione sistemica. Gli studi per identificare un fungo ospite del micovirus, responsabile della CCRS, non hanno portato a risultati conclusivi. Inaspettatamente però le indagini hanno rivelato, per analisi di rDNA e dei geni EF1-α e RBP1, la costante presenza del noto patogeno del ciliegio Taphrina wiesneri in tutti i campioni di foglie e di gemme analizzati, prelevati da ciliegi di varie aree della Campania e della Toscana, in ambienti diversi, seguiti in varie stagioni per 3 anni. Il fungo Taphrina risulta molto numeroso nelle gemme di ciliegio e perfino nelle strutture embrionali di teche polliniche. L’ibridazione in situ su sezioni di gemme di vari ciliegi e le colorazioni specifiche rivelano strutture fungine in posizioni non casuali. La numerosa ed organizzata presenza del fungo patogeno nelle strutture embrionali delle gemme e l’assenza di malattia, suggeriscono una forma di endofitosi, la prima riportata in alberi da frutto. Su una possibile endofitosi tra T. wiesneri e il ciliegio è in via di pubblicazione un lavoro accettato dal J. of Plant Pathology. In parallelo, negli ultimi due anni di dottorato, ho lavorato anche ad un progetto riguardante la eterogeneità genetica nei tessuti di un tumore primario del colon e di un tumore recidivo del colon-retto, di due pazienti. Di questa parte del lavoro non presento relazione. I risultati sono oggetto di un manoscritto (G. Geraci, I. D’Elia, R. del Gaudio and R. Di Giamo) inviato per la pubblicazione a PlosOne, che ha richiesto ulteriori dati che sono attualmente in elaborazione

Molecular evidence of Taphrina wiesneri in leaves and buds of healty sweet cherry (Prunus avium) A possible endophytism?

Analysis of DNAs from leaves and buds of several sweet cherry trees (Prunus avium) in two different Italian regions with different climates, has revealed the presence of Taphrina wiesneri, in all samples analyzed. The presence of other fungi was only occasional. The presence of T wiesneri was shown by sequencing segments of rDNA genes amplified by PCR. Analysis of genes coding for the elongation factor-1 alpha (EF1-alpha) and RNA-polymerase II subunit 1 (RPB1), confirmed the result. The T wiesneri sequences were variants of those reported in data bases. T wiesneri was not detected in buds of walnut, apricot and sour cherry growing close to the analysed sweet cherry trees. Determination of the relative numbers of T wiesneri and sweet cherry genomes in samples prepared from the same buds of 4 different trees all gave similar values indicating massive presence of the fungus. However, in situ hybridization experiments on bud sections using a T wiesneri-specific 18S rDNA probe and stains for fungi, labelled structures not clearly representing hyphae

Mutations in HPRT gene regions in tumour tissues of patient A, colorectum carcinoma relapse.

<p>Numeration of nucleotide positions of exons starts from A of the initial ATG triplet of the coding sequence. *, mutation already identified in cDNA samples from tumour tissues of patient B. <i>wt</i>: wild type sequence.</p

Mutations in TP53 transcripts in tumour and nearby healthy tissues.

<p>Numeration of nucleotide positions starts from A of the initial ATG triplet. <i>wt</i>: wild type sequence.</p

Mutations in G6PD transcripts in tumour and nearby healthy tissues.

<p>Mutations present in multiple clones in the same tissues are reported in bold-type, those present in different tissues are also underlined. °, mutation in healthy and tumour tissues of the same patient, +, mutation in the healthy tissues of both patients A and B. Numeration of nucleotide positions starts from A of the initial ATG triplet. <i>wt</i>: wild type sequence.</p

Mutation frequency values of all analyzed sequences of HPRT and G6PD cDNA and genes.

<p>Panel A, values of HPRT sequences; Panel B values of G6PD sequences. AH, AT, BH, BT: values calculated for cDNA clones of Tumour and nearby Healthy tissues of patients A and B. C1 and C2: values for cDNA clones from blood tissues of 2 healthy reference individuals. AT gene: values for clones of global genomic regions of DNA from tumour tissues of patient A. AT exons: values for the exon regions of the sequenced genomic regions. Controls: values for clone samples.</p

Percent mutated clones of HPRT and G6PD cDNA and genomic regions.

<p>Panel A, values of HPRT transcripts; Panel B, values of G6PD transcripts. Panel C, values of HPRT genomic regions; Panel D, values of G6PD genomic regions. Percent values have been calculated on pooled data from each independent analysis. AT and BT, values of clones of Tumour tissues of patients A and B, respectively; AH and BH, values of clones of Healthy tissues near tumours. C1 and C2, values of reference samples. Clones, values of control samples.</p

Clinical features of prognostic significance in myelodysplastic patients with normal karyotype at high risk of transformation

The International Prognostic Scoring System (IPSS) for myelodysplastic syndromes (MDS) has defined patients with a normal karyotype as a good risk cytogenetic subgroup, but nevertheless a fraction of these patients has a poor outcome similar to that of high risk patients. We retrospectively analysed our series of myelodysplastic patients with normal karyotype observed in a period of 11 years, with the aim of identifying clinical features of possible prognostic significance within this subgroup of patients. Multivariate analysis showed that among clinical scoring systems, the Bournemouth score appears the best prognostic indicator for risk of leukemic transformation, and platelet count &lt;100 × 10 9/l -1, presence of haemorrhagic symptoms at time of diagnosis and morphologic FAB classification are the main prognostic factors for prediction of survival. In the absence of genetic abnormalities as detected by conventional cytogenetics or even the more sensitive molecular techniques in MDS, clinical variables could be of help in identifying patients with different prognosis, suitable for risk adapted therapeutic strategies. © 2004 Elsevier Ltd. All rights reserved