MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 1013 vector genomes per milliliter (vg ml-1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection.

Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.

Comparison of DNA gains and losses in primary renal clear cell carcinomas and metastatic sites: importance of 1q and 3p copy number changes in metastatic events.

Archival material from primary and metastatic renal clear cell carcinomas of 25 patients was studied by comparative genomic hybridization. Copy number changes of entire chromosomes or chromosomal subregions were detected in 22 primary and 21 metastatic tumors. Copy number changes affected the following chromosomes in at least 20% of the 25 primary tumors (minimal common region given in parentheses): gains were noted for chromosomes 1 (1q21-->q23), 5 (5q31-->q34), 7 (7p), 8 (8q), 16 (16p), 17 (17q12-->qter), 19, and 22 (22q12-->qter); losses were revealed for chromosomes 3 (3p21-->pter), 8 (8p23-->pter), 14(14q21-->qter), and Y. The same chromosomal regions that were involved in primary renal clear cell carcinomas were also found in the respective metastatic tumors but with strikingly different frequencies for a few regions. Metastatic tumors showed a significantly higher frequency of complete or partial gains of the long arm of chromosome 1, in particular at 1q21-->q23 than primary tumors (16 cases versus 6 cases; P < 0.005). These data suggest a correlation of metastatic events in renal clear cell carcinomas with an increase in the copy number of genes located at 1q, in particular at 1q21-->q23. In contrast, the entire or partial loss of the short arm of chromosome 3 was significantly less frequent in metastatic tumors (8 cases versus 15 cases; P < 0.025). The validity of 1q and 3p copy number changes detected by comparative genomic hybridization was confirmed by interphase cytogenetics with region-specific yeast artificial chromosomes to paraffin-embedded tumor tissue sections.

Common regions of deletion in chromosome regions 3p12 and 3p14.2 in primary clear cell renal carcinomas.

Nearly all clear cell renal cell carcinomas (RCCs) exhibit loss of alleles on the short arm of chromosome 3. Loss and mutation at the von Hippel-Lindau (VHL) gene at 3p25 probably occurs in most RCCs and, since the VHL gene was recently cloned, data on VHL involvement in RCCs is accumulating. However, the region 3p14-p12, a region that contains the familial RCC-associated t(3;8)(p14.2;q24) chromosome translocation and the small cell lung carcinoma-associated homozygous deletion at 3p13-12, has also been reported to exhibit allele loss in a large fraction of RCCs. In order to focus future studies on potential suppressor genes in the 3p14-p12 region, we have studied allele loss in 30 RCCs with 9 polymorphic simple sequence repeat markers spanning 3p21.1-p12. Partial losses in the 3p21-p12 region were observed, allowing determination of common regions of loss of heterozygosity overlap in 15 RCCs. Results suggested that most RCCs exhibit loss in a region which brackets the t(3;8) familial chromosome translocation at 3p14.2, and some show additional deletions within the U2020 small cell lung carcinoma deletion at 3p12.

Absence or reduction of Fhit expression in most clear cell renal carcinomas.

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.

Loss of heterozygosity at the familial RCC t(3;8) locus in most clear cell renal carcinomas.

Previously, we had observed that more than 80% of clear cell renal carcinomas (RCCs) exhibited loss of heterozygosity (LOH) between the microsatellite markers D3S1285 (in 3p14.1) and D3S1295 (in 3p21.1), a region which includes the protein tyrosine phosphatase gamma locus (PTPRG locus, PTP gamma gene) and the 3p14.2 break of the familial RCC-associated translocation, t(3;8)(p14.2;q24), which has been hypothesized to affect expression of an RCC suppressor gene or oncogene. Using seven microsatellite markers and four markers derived from a PTPRG YAC contig, we have further delineated the 3p14.2 region of LOH in RCCs. Eighty-nine % of clear cell RCCs (31 of 35) showed a common region of loss between the D3S1481 and D3S1312 loci which flank the 3p14.2 t(3;8) translocation breakpoint and the PTP gamma gene. The PTP gamma gene occupies approximately 780 kilobase pairs between markers D3S1480 and D3S1312, with its currently defined 5' end greater than 200 kilobase pairs centromeric to the 3p14.2 translocation break. Although most of the RCCs with LOH between D3S1481 and D3S1312 loci have lost at least a portion of one PTP gamma allele, we have tested all known exons of the remaining PTP gamma gene in a number of the kidney tumors and have not observed mutations. Thus, there may be another gene in the vicinity of the 3p14.2 break that is important not only in the familial RCCs in the t(3;8) family but in the majority of clear cell RCCs.

Structure and expression of the human FHIT gene in normal and tumor cells.

The FHIT gene, encoded by 10 exons in a 1.1-kb transcript, encompasses approximately 1 Mb of genomic DNA, which includes the hereditary RCC t(3;8) translocation break at 3p14.2, the FRA3B common fragile region, and homozygous deletions in various cancer-derived cell lines. Because some of these genetic landmarks (e.g., the t(3;8) break between untranslated FHIT exons 3 and 4, a major fragile region that includes a viral integration site between exons 4 and 5, and cancer cell homozygous deletions in intron 5) do not necessarily affect coding exons and yet apparently affect expression of the gene product, we examined the FHIT locus and its expression in detail in more than 10 tumor-derived cell lines to clarify mechanisms underlying aberrant expression. We observed some cell lines with apparently continuous large homozygous deletions, which included one or more coding exons; cell lines with discontinuous deletions, some of which included or excluded coding exons; and cell lines that exhibited heterozygous and/or homozygous deletions, by Southern blot analysis for the presence of specific exons. Most of the cell lines that exhibited genomic alterations showed alteration of FHIT transcripts and absence or diminution of Fhit protein.

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes.

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing.

Detection of germline mutations in the BRCA1 gene by RNA-based sequencing.

BRCA1 mutation detection is expensive and has sensitivity limitations, which might at least partially be overcome by RNA-based sequencing. There are claims that RNA tests are unreliable due to differential splicing, exon skipping, or nonsense-mediated mRNA decay that results in either the absence or low expression of mRNA harboring mutations. The major aim of this study was to determine if the application of specific high temperature annealing primers can assure high sensitivity of detection of BRCA1 sequence alterations by cDNA sequencing. The study group comprised 21 Polish cancer families with aggregations of breast and/or ovarian cancer. We detected mutations in 10 out of 21 unrelated patients. These were: nucleotide substitutions (c.309T>C; c.300T>G); nucleotide insertions (c.5382insC) three cases; nucleotide deletions (c.4154delA) one case, (c. 185delAG) one case, (c.3819delGTAAA) two cases; and the deletion of the entire sequence of exon 22, one case. In addition, we identified three transcript variants resulting from alternative splice sites affecting the last six nucleotides of exon 1a (GTAAAG), and the first three nucleotides (CAG) of exon 8 and exon 14. In all cases these were cDNA heterozygous changes. Two of these splice site changes have not been previously described. Sequencing of genomic DNA "exon by exon" did not result in the detection of any additional abnormalities. The sensitivity of our analyses was sufficient to reliably detect mutations without the necessity of tissue culturing to obtain enough template cDNA for analysis.

Loss or reduction of Fhit expression in renal neoplasias: correlation with histogenic class.

Involvement of the 3p14.2 region of chromosome 3 in kidney cancers was suggested 20 years ago, when a reciprocal constitutional translocation, t(3;8)(p14.2;q24), was shown to segregate with bilateral clear cell renal carcinoma in 3 generations of 1 family. The FHITgene that is interrupted at 3p14.2 by the t(3;8) translocation has been isolated, characterized, and shown to be frequently altered, mainly by internal deletion, in carcinomas or cancer-derived cell lines of the lung, stomach, pancreas, esophagus, cervix, and colon. Although up to 90% of sporadic clear cell renal carcinomas, representing 70% of adult renal carcinomas, exhibit loss of FHIT alleles, FHIT gene alterations have been documented for only a few renal cell carcinoma-derived cell lines. Nevertheless, more than 50% of clear cell carcinomas were recently shown to express little or no Fhit protein, unlike the normal kidney tubule epithelium, which is uniformly strongly positive for Fhit expression. We have extended our immunohistochemical study of expression of Fhit protein to the spectrum of histopathologic subtypes of adult renal tumors. There is an apparent continuum of Fhit expression from the 100% strongly positive oncocytomas through mostly positive papillary and chromophobe to the mostly negative clear cell and sarcomatoid to the negative or predominantly negative collecting duct renal carcinomas. This pattern of diminishing Fhit expression correlates with reported frequency of 3p allele loss in renal carcinomas and may parallel the potential for aggressive behavior of tumors, as suggested by the abundant Fhit expression in the benign oncocytomas and the near absence of Fhit expression in sarcomatoid and collecting duct RCCs.

Losses at 3p common deletion sites in subtypes of kidney tumours: histopathological correlations.

Deletions of the short arm of chromosome 3 (3p) have been recognized as characteristic features of clear cell renal cell carcinomas (clear cell RCC). We analysed 55 clear-cell RCCs and 30 non-clear-cell kidney tumours (10 papillary and 7 chromophobic RCCs, 11 oncocytomas and 2 collecting duct carcinomas) in loss of heterozygosity (LOH) studies using microsatellite markers for previously observed regions of common deletions on 3p in kidney tumours (3p25, 3p21.3, 3p14.2 and 3p12-13). Alterations were found in all 55 cases of clear-cell RCCs at two to four of the 3p regions. Extensive losses were not found in non-clear-cell tumours except for collecting duct carcinomas; 1 of 10 papillary RCCs showed interstitial deletion limited to a single 3p21.3 locus. LOH analyses using microsatellite markers for regions of common deletions at 3p may be of value in differential diagnosis of kidney tumours.

Fhit expression is absent or reduced in a subset of primary head and neck cancer.

BACKGROUND: The inactivation of the FHIT gene at 3p14.2 by various mechanisms might be of importance in head and neck squamous cell carcinoma (HNSCC). Most reports are based on DNA and RNA findings of intragenic deletions and abnormal transcripts. MATERIAL AND METHODS: To study the protein expression of this putative tumour suppressor gene, we analysed 48 HNSCCs by immunohistochemistry using a polyclonal antibody (ZR44). The results were compared with mutation analysis, clinical data and loss of heterozygosity (LOH) data at 3p14.2. RESULTS: Complete absence of Fhit expression was detected in 8 out of 48 of tumours (17%) and 3 tumours (6%) showed heterogenous staining. The overall frequency of LOH for microsatellite D3S1234 was 64% and 5/7 of Fhit negative tumours exhibited LOH. CONCLUSION: Our findings provide further evidence that FHIT is inactivated in a subtype of HNSCC; however, the incidence of lack of Fhit expression compared to the high frequency of LOH on chromosome 3p supports the notion of additional tumour suppressor genes at 3p14.

Non-isotopic procedure with silver staining of polyacrylamide gels in detection of genomic abnormalities in tumors using microsatellites.

Microsatellite-based PCR (polymerase chain reaction) with silver staining of polyacrylamide gels as an alternative approach to detect genomic abnormalities in tumors has been developed. In experiments with a series of primers for microsatellite sequences located on human chromosome 5 we found that a 2- to 4-fold increase or decrease in the ratio of amount of allelic microsatellite sequences can be reliably assessed from the relative intensity of corresponding PCR products. Results of the studies of renal carcinoma cell lines carrying increased or decreased numbers of specific allelic markers on chromosome 5 assessed by cytogenetics, Southern blotting-restriction fragment length polymorphism and microsatellite analyses were consistent. Microsatellite studies performed on samples obtained from formalin-fixed paraffin-embedded archival material allowed detection of genomic abnormalities of chromosome 5 in five of ten primary renal cell carcinomas.

Fhit protein expression in endometrial cancers: no correlation with histological grade.

The genes specifically involved in endometrial cancers have not yet been discovered. The FHIT gene, a tumour suppressor located at 3p14.2, is altered in many human tumours, including those derived from the female genital tract. We have thus investigated the status of Fhit protein expression in endometrial carcinomas (EC), and its association with histological grade of malignancy in order to determine if Fhit expression is inactivated in EC and if so, whether it is inactivated during initiation or progression. Recent studies have reported that alteration in the FHIT locus detected by DNA and RNA analysis is well correlated with loss of Fhit protein expression in tumours. Thus, we characterised Fhit protein expression as an indication of FHIT gene status in 35 cases of EC of different histological grade (G1: 13 cases; G2: 14 cases; G3: 8 cases). In our group of cancers, Fhit protein expression was absent or reduced in 37% (13/35) of EC. The first 13 cases, judged as G1, showed Fhit deficiency in approximately 38.5% of cases (5/13). For G2 and G3 tumours these numbers were similar and accounted for approximately 35.7% (5/14) and approximately 37.5% (3/8), respectively. No statistical difference was found for Fhit expression among the various groups of tumours, which allowed us to conclude that morphological grade does not seem to be an important factor. Our results suggest that Fhit inactivation is an early event in carcinogenesis of the endometrium. As this observation is contrary to some already published reports, another independent study with larger amounts of material is necessary to determine this issue definitely.

Fhit protein expression in hereditary and sporadic colorectal cancers.

The majority of hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in DNA mismatch repair genes, especially in MLH1 and MSH2. Tumours in such patients also show microsatellite instability characteristic for DNA repair defects. The FHIT gene, a candidate tumour suppressor gene located at 3p14.2 has been shown to be involved in carcinogenesis of many human tissues, including digestive tract tissues. In our study, we characterized Fhit protein expression in hereditary and sporadic colorectal cancers (CRC). Our intention was to determine if cancers with mutations in the mismatch repair genes, MSH2 and MLH1, would show more frequent inactivation of the FHIT gene. Sixteen HNPCC and 28 sporadic CRC cases were examined by standard immunohistochemical analyses. Both study groups comprised carefully and selectively chosen cases. We have observed higher frequency of loss or reduction of Fhit protein expression in hereditary CRC than in sporadic cases (44% vs. 25%). Although this difference was not statistically significant (p = 0.17), possibly due to the small number of available tumour specimens, the tendency is interesting. More extensive studies on a larger number of cases should be done in the HNPCC group to confirm statistical significance. Our results suggest that the FHIT gene plays an important role in carcinogenesis of at least one fourth of all colorectal cancers.

Environmental factors may regulate BCL2 associated lymphomagenesis: a very low incidence of BCL2-MBR translocation in Poland.

A characteristic feature of follicular non-Hodgkin's lymphomas (FL) is a chromosome translocation t(14;18)(q32;q21). In American patients the t(14;18) can be found in the large majority (approximately 70%) of FL and in a significant (10-40%) percentage of diffuse large cell lymphomas (DLCL). However there are reports suggesting that geographic or racial factors may regulate genesis of lymphomas with BCL2 abnormalities. Herein we present results of molecular analysis for t(14;18) in lymphomas in Poland. Analyses were performed on 37 cases of FL, 55 cases of diffuse lymphomas (DL) and 39 cases of Hodgkin's disease (HD). By Southern and polymerase chain reaction BCL2 translocation within major breakpoint region was found only in 3 of 37 FL, 1 of 55 DL and 2 of 39 HD. These results are the lowest reported ratio. We hypothesize that environmental factors may regulate BCL2 associated lymphomagenesis.

[HPV virus infections and other promotion factors of the carcinogenic process in girls]. / Zakazenie wirusem HPV oraz inne czynniki promocyjne w procesie karcinogenezy u dziewczat.

HPV virus infections and other promotion factors in the development of carcinogenic process at 101 girls from different social groups were evaluated. Chronic infection of the cervix was found at the group of 56 smoking girls, aged 15-18, having sexual contacts with many partners. 10 of them were infected by HPV virus. These data indicate that particular health care and widely understood cervical carcinoma prophylaxis in such groups are necessary.

[Detection of tumor virus infections with HPV 16, 18, and 33 from cytologic material in the uterine neck using the PCR method]. / Wykrywanie infekcji wirusami HPV 16, 18 i 33 w materiale cytologicznym z szyjki macicy metoda PCR.

A method for detection of oncogenic viruses HPV 16, 18, 33 based on enzymatic amplification (PCR) of specific viral DNA sequences is described. This technique has many advantages, including: specificity, reproducibility, relatively simple procedure and low cost.

Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders.

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.